Multi-specific polypeptide constructs with restricted CD3 binding and methods of use thereof

Abstract

The present application relates to multispecific polypeptide constructs with restricted CD3 binding and methods of use thereof. The present invention relates generally to multispecific polypeptides with restricted CD3 binding. In some embodiments, the multispecific polypeptide contains a cleavable linker that, upon cleavage, produces a dual effector function. Also provided are methods of making such multispecific polypeptides and methods of using such multispecific polypeptides in a variety of therapeutic, diagnostic, and prophylactic indications.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880038526.5.

[0002] This application claims priority to U.S. Provisional Application No. 62 / 484,217, filed April 11, 2017, entitled “Multispecific Polypeptide Constructs with Restricted CD3 Binding and Methods of Use Thereof,” the entire contents of which are incorporated herein by reference.

[0003] The sequence listing is incorporated by reference

[0004] This application is filed in electronic format with a sequence listing. The sequence listing is provided as a file titled 744952000141SeqList.TXT, created on April 11, 2018, and 174,179 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Technical Field

[0005] The present invention generally relates to multispecific polypeptides with restricted CD3 binding. In some embodiments, the multispecific polypeptides contain a cleavable linker that, upon cleavage, generates dual effector functions. Also provided are methods for preparing such multispecific polypeptides and methods for using such multispecific polypeptides for a variety of therapeutic, diagnostic, and prophylactic indications. Existing technology

[0006] Therapeutic antibodies that lead to target cell depletion generally rely on effector functions mediated by interactions with Fc-γ-receptors (FcγRs) and complement proteins. Effector cells expressing FcγRs are primarily those of the innate immune system. T cells are not the direct effector cells involved in antibody-mediated target cell depletion.

[0007] The CD3 (cluster of differentiation 3) T cell coreceptor is a multimeric protein composed of four different polypeptide chains, designated the ε, γ, δ, and ζ chains. The CD3 complex serves as the signaling module of the T cell receptor (TCR) that non-covalently associates with the antigen-binding a / b chains of the T cell receptor (TCR).

[0008] Since direct engagement of CD3 leads to T cell activation, it is a desirable target for a variety of therapeutic and / or diagnostic indications. Therefore, there is a need for antibodies and therapeutic agents that target the CD3 / TCR pathway. Summary of the Invention

[0009] The present invention provides multispecific polypeptide constructs that exhibit limited CD3 binding. This is due to this aspect of such cytokines. In some embodiments, the multispecific polypeptide construct is composed of a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled or operably connected by a linker, wherein the Fc region is located at the N-terminus of the CD3 binding region; and one or both of the first and second components comprise an antigen binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct in an inactive state is composed of a first component and a second component, wherein the first and second components are operably connected, wherein each of the first and second components comprises an antigen binding domain that binds to a tumor-associated antigen (TAA), wherein the first component comprises an Fc region, wherein the second component comprises a CD3 binding region, and wherein the first and second components are coupled by a cleavable linker. In some embodiments, the CD3 binding region binds to CD3 (CD3ε).

[0010] In some embodiments, the antigen binding domain is located at the amino terminus of the Fc region of the multispecific polypeptide construct and / or at the carboxyl terminus relative to the CD3 binding region. In some embodiments, the first component comprises a first antigen binding domain and the second component comprises a second antigen binding domain, wherein each of the antigen binding domains binds to a tumor associated antigen (TAA). In some cases, the first antigen binding domain is located at the amino terminus of the multispecific construct and the second antigen binding domain is located at the carboxyl terminus of the multispecific construct. In some embodiments, the first antigen binding domain is located at the amino terminus of the Fc region of the multispecific polypeptide construct and / or at the carboxyl terminus relative to the CD3 binding region.

[0011] Provided herein are multispecific polypeptide constructs, wherein the multispecific construct comprises, in order from N-terminus to C-terminus: a first antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3-binding region that binds CD3 (CD3ε); and a second antigen-binding domain that binds to a tumor-associated antigen (TAA). Also provided are multispecific polypeptide constructs, wherein the multispecific construct comprises, in order from N-terminus to C-terminus: an immunoglobulin Fc region; a linker; a CD3-binding region that binds CD3 (CD3ε); and an antigen-binding domain that binds to a tumor-associated antigen (TAA). Provided herein are multispecific polypeptide constructs, wherein the multispecific construct comprises, in order from N-terminus to C-terminus: an antigen-binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3-binding region that binds CD3 (CD3ε).

[0012] The present invention provides multispecific polypeptide constructs that bind at least CD3 and a second antigen, such as a tumor-associated antigen (TAA). The multispecific polypeptide constructs provided herein include at least a first component comprising one or more copies of an antigen-binding domain that binds an antigen linked to the Fc region of an immunoglobulin; a second component comprising one or more copies of at least one binding domain that binds CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding region, which are used interchangeably herein); and a linker, such as a cleavable linker, connecting the first and second components.

[0013] Positioning the Fc region N-terminally to the CD3 binding domain reduces or prevents the CD3 binding domain's ability to bind CD3. In some embodiments, in the uncleaved / inactive state, the first component (component 1) and the second component (component 2) of the multispecific polypeptide construct are linked and do not permit binding to CD3 unless the antigen-binding domain is bound to its cognate antigen. This is advantageous because it prevents the CD3 binding domain from systemically binding to T cells and localizes it to sites of antigen expression. This is beneficial because it eliminates the primary binding groove for peripheral T cells, thereby allowing for more favorable distribution and localization at sites of antigen expression (e.g., tumor cells or the tumor microenvironment). In some cases, CD3 binding and / or engagement can be amplified or increased by including a cleavable linker connecting component 1 and component 2, wherein upon cleavage of the cleavable linker (e.g., by proteolysis), binding by the CD3 binding domain is increased.

[0014] In the inactive, i.e., uncleaved, state, components 1 and 2 of the multispecific polypeptide construct are operably linked to CD3 and do not bind or otherwise engage CD3 unless the antigen-binding domain is bound to its cognate antigen. In some embodiments, the uncleaved multispecific polypeptide construct can interact with FcγRs and mediate innate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the uncleaved multispecific polypeptide construct can interact with complement proteins (i.e., C1q) and mediate complement-dependent cytotoxicity.

[0015] The multispecific polypeptide constructs of the present disclosure typically have more than one antigen-binding domain. In the aspects provided wherein the multispecific polypeptide construct contains a cleavable linker, once the linker connecting the first and second components is cleaved by, for example, a protease, each component retains at least one antigen-binding domain. The first component (i.e., component 1) contains at least an Fc region and an antigen-binding domain. The second component (i.e., component 2) contains at least an anti-CD3 binding domain and an antigen-binding domain.

[0016] Cleavage within the cleavable linker (e.g., by proteolytic cleavage) physically separates component 1 from component 2, each of which has therapeutic uses, but depends on different effector cells. Component 1 contains at least one antigen-binding domain and an Fc region. In some embodiments, component 1 is capable of inducing innate immune effector functions, such as ADCC, cytokine release, degranulation, and / or phagocytosis. Component 2 contains at least a CD3 binding domain and an antigen-binding domain, the former capable of binding CD3 (when separated from component 1). Component 2 is capable of forming an immunological synapse between antigen-expressing cells and T cells. This co-engagement mediates antigen-dependent T cell activation, cytotoxicity, cytokine release, degranulation, and proliferation. In the cleaved / activated state, component 2 is not operably linked to the Fc region of component 1 and, as such, cannot interact with FcRn. If localized to sites devoid of antigen-expressing cells, component 2 has enhanced serum clearance. This is advantageous because it limits systemic exposure of the activating anti-CD3 binding domain and directly localizes it to antigen-expressing tissues (e.g., tumor cells or the tumor microenvironment).

[0017] In some embodiments, the multispecific polypeptide is in an inactive state, i.e., uncleaved, and binding of the CD3 binding region to CD3 is inhibited or significantly reduced when the multispecific polypeptide construct is in the uncleaved state compared to the cleaved state. In some embodiments, the multispecific polypeptide is in an activated state, and the first and second components are not operably linked. In some embodiments, the multispecific polypeptide is in an activated state, i.e., cleaved, and the second component binds to the epsilon chain of CD3 (CD3ε) and a tumor-associated antigen (TAA).

[0018] In some aspects, the antigen binding domain, or each antigen binding domain independently, is selected from an antibody or antigen binding fragment, a natural cognate binding partner, anticalin (engineered lipocalin), darpin, fynomer, centyrin (engineered fibronectin III domain), cysteine-knob domain, affilin, affibody or an engineered CH3 domain. In some embodiments, the natural cognate binding partner comprises the extracellular domain of a natural cognate binding partner of a TAA or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA.

[0019] In some aspects, the antigen binding domain, or independently each antigen binding domain, comprises the extracellular domain of a natural cognate binding partner of a TAA, or a binding fragment thereof, or a variant thereof that exhibits binding activity for a TAA.

[0020] In some embodiments, the first component comprises one or more copies of an antigen binding domain. In some embodiments, the first component comprises at least two antigen binding domains, such as two antigen binding domains. In some embodiments, at least two antigen binding domains of the first component bind to the same TAA. In some cases, at least two antigen binding domains of the first component bind to different epitopes on the same TAA. In some cases, at least two antigen binding domains of the first component bind to the same epitope of the same TAA. In some embodiments, at least two antigen binding domains of the first component bind to different TAAs.

[0021] In some embodiments, the antigen binding domain of the first component, which in some cases is the first antigen binding domain, comprises one or more copies of an antibody or an antigen binding fragment thereof. In some embodiments, the antigen binding domain of the first component, such as the first antigen binding domain, comprises one or more copies of an antibody or an antigen binding fragment thereof selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the first antigen binding domain comprises one or more single domain antibody (sdAb) fragments (e.g., V H H, V NAR , after transformation V H or V K One or more copies of a domain). H H can be generated from camelid heavy chain only antibodies. V NAR Heavy chain antibodies can be generated from cartilaginous fish only. Various methods have been performed to generate heterodimeric V H and V K Domain-based generation of monomeric sdAbs involves surface engineering and selection of specific germline families.

[0022] In some embodiments, the antigen binding domain of the first component, such as the first antigen binding domain, binds to an antigen, such as a tumor associated antigen (TAA). In some embodiments, the TAA is selected from the group consisting of 1-92-LFA-3, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, C D44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD12 3. CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEA CAM6 (NCA-90), claudin-3, claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, I L21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine phosphate 1, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4 , TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2 and WISP-3.

[0023] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region.

[0024] In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 subclasses. In some embodiments, the Fc region is an Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof. In some embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some cases, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2. In some of any such embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.

[0025] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

[0026] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications to prevent glycosylation, alter Fc receptor interactions, reduce Fc receptor binding, enhance interaction with CD32A, reduce complement protein C1q binding, extend half-life, enhance FcRn binding, alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize CH3:CH3 surface homodimerization, and combinations thereof.

[0027] In some embodiments, the Fc is a heterodimeric Fc. In some cases, one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization compared to a polypeptide of the homodimeric Fc region, optionally compared to an Fc polypeptide set forth in SEQ ID NO: 1 or an immunologically active fragment thereof. In some embodiments, each of the Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification. In some cases, each of the Fc polypeptides of the heterodimeric Fc comprises a knob-into-hole modification or comprises a charge mutation to increase electrostatic complementarity of the polypeptides. In some embodiments, the amino acid modification is a knob-into-hole modification.

[0028] In some embodiments, the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr366Ser, Leu368Ala, Tyr407Val, and combinations thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W. In some cases, the first and second Fc polypeptides further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification of the first polypeptide is at one of positions Ser354 and Y349, and the modification of the second Fc polypeptide is at the other of positions Ser354 and Y349.

[0029] In some embodiments, the amino acid modification is a charge mutation to increase the electrostatic complementarity of the polypeptide. In some embodiments, the first and / or second Fc polypeptide comprises a modification of a complementary position, wherein the modification is the replacement of the complementary amino acid of the other polypeptide with an amino acid having an opposite charge. In some embodiments, the first or second polypeptide comprises a modification of a complementary position, wherein the modification is the replacement of the complementary amino acid of the other polypeptide with an amino acid having an opposite charge. In some embodiments, at least the first or second Fc polypeptide each comprises a modification of a complementary position, wherein the modification is the replacement of the complementary amino acid of the other polypeptide with an amino acid having an opposite charge. In some embodiments, the first and second Fc polypeptide each comprises a modification of a complementary position, wherein the modification is the replacement of the complementary amino acid of the other polypeptide with an amino acid having an opposite charge.

[0030] In some embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification of residue Ile253. In some cases, the modification is Ile253Arg. In some embodiments, one of the first or second Fc polypeptides of the heterodimeric Fc further comprises a modification of residue His435. In some cases, the modification is His435Arg. In some embodiments, the Fc region comprises a polypeptide without Lys447.

[0031] In some embodiments, modifications within the Fc region reduce binding to the Fc-receptor-gamma receptor, but have minimal effect on binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutated or modified Fc polypeptide comprises the following mutations: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V) using the Kabat numbering system.

[0032] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding. In some embodiments, the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof. In some cases, the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof. In some specific embodiments, the modifications are at positions Met252 and Met428. In some cases, the modifications are Met252Y and Met428L. In some cases, the modifications are Met252Y and Met428V.

[0033] In some embodiments, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 82, 86, 94, or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 83, 87, 90, 92, 98, or 100.

[0034] In some embodiments, the Fc region comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some aspects, the first polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 84, 88, 95, or 97, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 85, 89, 91, 93, 99, or 101.

[0035] In some embodiments, the Fc region comprises a polypeptide comprising at least one modification to enhance FcγR binding. In some cases, the modification is modification of Ser239 or Ile332. In some embodiments, the glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region. In some embodiments, the Fc region has no or reduced fucose content.

[0036] In some embodiments, the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL). In some of any such embodiments, the CD3 binding region is monovalent.

[0037] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, optionally not a single-chain variable fragment (scFv). In some embodiments, the Fc is a heterodimeric Fc and comprises the VH and VL of the anti-CD3 antibody or antigen-binding fragment connected to opposite polypeptides of the heterodimeric Fc. In some embodiments, the CD3 binding region is unable or substantially unable to bind or engage CD3 unless at least one of the antigen-binding domains is bound to its TAA. In some aspects, the CD3 binding region is unable or substantially unable to bind or engage CD3 unless at least two of the antigen-binding domains are bound to their TAA.

[0038] In some embodiments, the multispecific polypeptide construct contains a linker that is a polypeptide linker. In some embodiments, the linker is a polypeptide of up to 25 amino acids in length. In some cases, the linker is or is about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 In some embodiments, the linker is a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some cases, the linker is a cleavable linker.

[0039] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, the linker within such components is primarily composed of the amino acids glycine and serine, denoted herein as a GS-linker. The GS-linkers of the fusion proteins of the present invention have varying lengths, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0040] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, also known as (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, also known as (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, also known as (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, also known as (GGS)5 (SEQ ID NO: 13).

[0041] In some embodiments, the second component also includes one or more copies of an anti-CD3 binding domain. In some embodiments, the anti-CD3 binding domain includes one or more copies of an antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD3 binding domain includes one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the anti-CD3 binding domain includes an Fv antibody fragment that binds to CD3ε (referred to herein as an anti-CD3ε Fv fragment). In some embodiments, the anti-CD3ε Fv antibody fragment includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0042] In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0043] In some embodiments, the second component comprises one or more copies of an antigen binding domain. In some embodiments, the second component comprises at least two antigen binding domains, such as two antigen binding domains. In some embodiments, at least two antigen binding domains of the second component bind to the same TAA. In some cases, at least two antigen binding domains of the second component bind to different epitopes on the same TAA. In some embodiments, at least two antigen binding domains of the second component bind to different TAAs.

[0044] In some embodiments, the first component contains a first antigen binding domain and the antigen binding domain of the second component is a second antigen binding domain. In some embodiments, the second antigen binding domain of the second component binds to the same antigen as the first antigen binding domain of the first component. In some embodiments, the second antigen binding domain of the second component binds to a different epitope on the same antigen as the first antigen binding domain of the first component. In some embodiments, the second antigen binding domain of the second component binds to an epitope on the same antigen as the first antigen binding domain of the first component.

[0045] In some embodiments, the antigen binding domain of the second component, such as the second antigen binding domain, comprises one or more copies of an antibody or an antigen binding fragment thereof. In some embodiments, the second antigen binding domain comprises one or more copies of an antibody or an antigen binding fragment thereof selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the second antigen binding domain comprises one or more single domain antibody (sdAb) fragments (e.g., V H H, V NAR , after transformation V H or V K One or more copies of a domain). H H can be generated from camelid heavy chain only antibodies. V NAR Heavy chain antibodies can be generated from cartilaginous fish only. Various methods have been performed to generate heterodimeric V H and V K Domain-based generation of monomeric sdAbs involves surface engineering and selection of specific germline families.

[0046] In some embodiments, the antigen binding domain of the second component, such as the second antigen binding domain, binds to an antigen, such as a tumor associated antigen (TAA). In some embodiments, the TAA is selected from the group consisting of 1-92-LFA-3, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, april J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD 41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXC L10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GL UT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nacastroin, Notch receptor, Notch1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine phosphate 1, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4 , TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0047] In some embodiments, the second antigen binding domain and the anti-CD3 binding domain are operably linked via an amino acid linker. In some embodiments, the linker within such components is primarily composed of the amino acids glycine and serine, denoted herein as a GS-linker. The GS-linker of the fusion protein of the present invention can have varying lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0048] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, also known as (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, also known as (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, also known as (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, also known as (GGS)5 (SEQ ID NO: 13).

[0049] Provided herein are multispecific polypeptide constructs comprising a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL), wherein: the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are opposite polypeptides linked to the heterodimeric Fc; the first and second components are coupled by a cleavable linker, wherein the heterodimeric Fc region is located at the N-terminus of the anti-CD3 antibody; and one or both of the first and second components comprise an antigen-binding domain that binds a tumor-associated antigen (TAA).

[0050] In some embodiments, the binding of the CD3 binding region to CD3 is significantly reduced when the multispecific polypeptide construct is in an uncleaved state compared to a cleaved state. In some embodiments, in the cleaved state, the first and second components are not linked.

[0051] In some embodiments, the cleavable linker is a polypeptide. In some embodiments, the cleavable linker is a polypeptide that is a substrate for a protease. In some embodiments, the protease is produced by immune effector cells, by tumors, or by cells present in the tumor microenvironment. In some embodiments, the protease is produced by a tumor near a cell expressing CD3ε and / or by a tumor co-localized with cells expressing CD3ε in a tissue, and wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by a tumor near a cell expressing one or more tumor-associated antigens (TAA) and / or by a tumor co-localized with cells expressing the target TAA in a tissue, and wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by an immune effector cell. In some embodiments, the protease is produced by an immune effector cell near a cell expressing a TAA. In some embodiments, the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NKT cell. In some embodiments, upon exposure of the multispecific polypeptide construct to a protease, the protease cleaves a cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by an immune effector cell in proximity to a cell expressing a TAA, and wherein upon exposure of the multispecific polypeptide construct to the protease, the protease cleaves a cleavable linker in the multispecific polypeptide construct.

[0052] In some embodiments, the cleavable linker is a polypeptide having a length of up to 50 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 25 amino acids. In some embodiments, the cleavable linker is a polypeptide having a length of up to 15 amino acids.

[0053] In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of proteases described herein. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, asparagine endopeptidase, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and any combination thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, asparagine endopeptidase, and matriptase. In some embodiments, the protease is selected from matriptase, matrix metalloproteinase (MMP), granzyme B, and a combination thereof.

[0054] In some embodiments, the protease is granzyme B. In some embodiments, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓P1' (SEQ ID NO: 150), wherein P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A. In some embodiments, the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P 1 ↓P1' (SEQ ID NO: 151), wherein P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G. In some embodiments, the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO: 136), LEPDG (SEQ ID NO: 152), LEADT (SEQ ID NO: 137), IEPDG (SEQ ID NO: 138), IEPDV (SEQ ID NO: 139), IEPDS (SEQ ID NO: 140), IEPDT (SEQ ID NO: 141), or LEADG (SEQ ID NO: 153). In some cases, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 105-112, 136-141, 148, 150-153.

[0055] In some embodiments, the protease is a mitogen-activated protein kinase. In some cases, the cleavable linker comprises the sequence P1QAR↓(A / V) (SEQ ID NO: 154), wherein P1 is any amino acid; or the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155). In some embodiments, the cleavable linker comprises the sequence RQARV (SEQ ID NO: 156). In some cases, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 154-156.

[0056] In some embodiments, the protease is a MMP. In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker comprises the formula P3P2P1↓P1' (SEQ ID NO: 157), wherein P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1' is L, I, or M. In some cases, the cleavable linker comprises the formula P3P2P1↓P1' (SEQ ID NO: 158), wherein P3 is P; P2 is Q or D; P1 is A or N; and P1' is L or I. In some embodiments, the cleavable linker comprises the sequence PAGL (SEQ ID NO: 24). In some embodiments, the cleavable linker is a substrate for a matrix metalloprotease (MMP).

[0057] In some embodiments, the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first and second polypeptides comprise at least one antigen-binding domain that binds to a tumor-associated antigen (TAA). In some cases, only one of the first or second polypeptide comprises at least one antigen-binding domain that binds to a TAA.

[0058] In some of any of the embodiments provided, the antigen binding domains produce monovalent, bivalent, trivalent, or tetravalent binding to a TAA. In some embodiments, one or more TAA-binding antigen binding domains are independently selected from sdAb, scFv, or Fab. In some embodiments, one or more TAA-binding antigen binding domains are TAAs that are single-chain molecules, such as single-chain antibody fragments containing VH and VL, such as sdAb or scFv. In some embodiments, at least one of the antigen binding domains is a Fab containing a first chain comprising VH-CH1 (Fd) and a second chain comprising VL-CL.

[0059] In some embodiments, at least one antigen binding domain is located amino-terminal to the Fc region and / or carboxyl-terminal to the CD3 binding region of one of the first or second polypeptide of the multispecific polypeptide construct. In some cases, at least one antigen binding domain is located amino-terminal to the Fc region of the multispecific construct, and the second antigen binding domain is located carboxyl-terminal to the CD3 binding region of the multispecific construct.

[0060] In some embodiments, at least one of the antigen-binding domains is a Fab. In some embodiments, the multispecific polypeptide construct comprises: (i) a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, and a VH domain of an anti-CD3 antibody or antigen-binding fragment; (ii) a second polypeptide comprising a second Fc polypeptide of a heterodimeric Fc region, a linker, and a VL domain of an anti-CD3 antibody or antigen-binding fragment, and (iii) a third polypeptide comprising the VH-CH1 (Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first and / or second polypeptide further comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some cases, only one of the first or second polypeptide comprises the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some embodiments, both the first or second polypeptide comprise the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some cases, the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is amino-terminal to the Fc region and / or carboxyl-terminal to the CD3 binding region of one of the first or second polypeptides of the multispecific polypeptide construct. In some embodiments, the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment is amino-terminal to the Fc region of the first or second polypeptide and carboxyl-terminal to the CD3 binding region of the other of the first or second polypeptide.

[0061] In some embodiments, the antigen binding domain, or each of the antigen binding domains independently, binds to a tumor antigen selected from the group consisting of: 1-92-LFA-3, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, april J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, C D40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL 10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-C SFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R,IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1=R, LAG=3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nacastroin protein, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX=40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1 GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine phosphate 1, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2 and WISP-3.

[0062] In some embodiments, the multispecific antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same TAA. In some cases, the first antigen-binding domain and the second antigen-binding domain bind to different epitopes on the same TAA. In some cases, the first antigen-binding domain and the second antigen-binding domain bind to the same epitope of the same TAA. In some embodiments, the multispecific antigen-binding domain comprises at least a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain bind to different TAAs.

[0063] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between a first antigen-binding domain and an immunoglobulin Fc polypeptide region (Fc region). In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between an anti-CD3 binding domain (CD3 binding region) and a second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between a first antigen-binding domain and an immunoglobulin Fc polypeptide region (Fc region), and a second connecting peptide (LP2) between an anti-CD3 binding domain (CD3 binding region) and a second antigen-binding domain.

[0064] In some embodiments, the uncleaved multispecific polypeptide construct has the following structural configuration from N-terminus to C-terminus: first antigen-binding domain - LP1 - immunoglobulin Fc polypeptide linker region (Fc region) - linker (e.g., cleavable linker) - anti-CD3 binding domain - LP2 - second antigen-binding domain. In some embodiments, the uncleaved multispecific polypeptide construct has the following structural configuration from N-terminus to C-terminus: second antigen-binding domain - LP2 - anti-CD3 binding domain (CD3 binding region) - linker (e.g., cleavable linker) - immunoglobulin Fc polypeptide linker region - LP1 - first antigen-binding domain. In some embodiments, the linker is a cleavable linker. In some embodiments, the two connecting peptides are different from each other. In some cases, LP1 or LP2 is independently a peptide of about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 independently comprises a peptide that is or comprises any of the Gly-Ser linkers shown in SEQ ID NOs: 10-13, 119, 135, 147, and 149.

[0065] In some embodiments, the multispecific construct is a Figure 1 In some embodiments, the multispecific construct is a construct having any of the structural configurations shown in Figure 2In some embodiments, the bispecific construct has the following structural configuration from the N-terminus to the C-terminus. The N-terminus of the bispecific construct includes a first antigen binding domain that binds to a tumor-associated antigen (TAA). The first binding domain binds to a first epitope on the TAA target. A central immunoglobulin Fc polypeptide region that regulates FcγR interactions and / or FcRn interactions is coupled to the first antigen binding domain. In some embodiments, the central immunoglobulin Fc polypeptide region is a heterodimer. The immunoglobulin Fc polypeptide region is coupled to a cleavable linker that contains one or more proteolytic cleavage sites located at the C-terminal position of the end of the immunoglobulin Fc polypeptide region. In some embodiments, the one or more proteolytic cleavage sites are substrates for interstitial proteases, matrix metalloproteinases (MMPs), or granzyme B. The cleavable linker is attached to the anti-CD3 binding sequence located at the C-terminus of the Fc and, in some cases, the distal end of the second component.

[0066] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is an Fv antibody fragment. In some embodiments, the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3 binding sequence is an Fv antibody fragment that has been engineered to include a disulfide bond between the variable heavy chain (VH) region and the variable light chain (VL) region, thereby generating a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the VH and VL domains comprising the anti-CD3 Fv are operably linked to opposing members of the heterodimeric Fc region. In such embodiments, the anti-CD3 Fv binds to CD3 in a monovalent manner. When the cleavable linker is intact, i.e., in an uncleaved or inactive state, the anti-CD3 dsFv does not bind to CD3. The C-terminus of the bispecific construct comprises a second antigen-binding domain that binds to a TAA. In some embodiments, the second antigen-binding domain binds to the same TAA as the first antigen-binding domain located within the first component. In some embodiments, the second antigen binding domain binds to a second epitope on the TAA, wherein the second epitope is non-competitive with the first epitope on the TAA. In some embodiments, the second antigen binding domain binds to a different TAA than the first antigen binding domain.

[0067] In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or an antigen-binding fragment thereof. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of: Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, the antigen-binding domain or each of the independently antigen-binding domains is selected from the group consisting of: Fab fragments, F(ab')2 fragments, Fv fragments, scFv, scAb, dAb, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, each of the first and second antigen-binding domains of the bispecific construct comprises one or more copies of a single domain antibody (sdAb) fragment (e.g., V H H, V NAR , after transformation V H or V K One or more copies of a domain). H H can be generated from natural camelid heavy chain only antibodies, genetically modified rodents producing heavy chain only antibodies, or naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR Heavy chain antibodies can be generated from cartilaginous fish only. Various methods have been performed to generate heterodimeric V H and V K Domain-based generation of monomeric sdAbs involves surface engineering and selection of specific germline families.

[0068] In some embodiments, the antibody or antigen binding fragment is an sdAb. In some cases, the sdAb is a human or humanized sdAb. In some aspects, the sdAb is a V H H, V NAR , a modified VH domain or a modified VK domain. In some embodiments, the antibody or its antigen-binding fragment is a scFv. In some cases, the antibody or its antigen-binding fragment is a Fab.

[0069] In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence of TYAMN (SEQ ID NO: 16); a VH CD2 comprising the amino acid sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 comprising the amino acid sequence of HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 comprising the amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 19); a VLCDR2 comprising the amino acid sequence of GTNCRAP (SEQ ID NO: 20); and a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 21).

[0070] In some embodiments, the anti-CD3 dsFv comprises: a VH having an amino acid sequence of any one of SEQ ID NOs: 14, 44, and 32-62, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14, 44, and 32-62; and a VL having an amino acid sequence of any one of SEQ ID NOs: 15, 72, and 63-81, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14, 44, and 32-62. In some instances, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15. In some instances, the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO:44 and the amino acid sequence of SEQ ID NO:72.

[0071] In some embodiments, the immunoglobulin Fc region of the first component is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0072] In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, comprising one or more modifications to prevent glycosylation, alter Fc receptor interactions, reduce Fc receptor binding, enhance interaction with CD32A, reduce complement protein C1q binding, extend half-life, enhance FcRn binding, alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), induce heterodimerization, prevent dimerization, stabilize CH3:CH3 surface homodimerization, and combinations thereof. In some embodiments, modifications within the Fc region reduce binding to the Fc-receptor-gamma receptor while having minimal effect on binding to the neonatal Fc receptor (FcRn). In some embodiments, the mutant or modified Fc polypeptide comprises the following mutations: using the Kabat numbering system, Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0073] In some embodiments, the first antigen-binding domain and the immunoglobulin Fc polypeptide are operably linked via an amino acid linker. In some embodiments, the linker within such components is primarily composed of the amino acids glycine and serine, denoted herein as a GS-linker. The GS-linker of the fusion protein of the present invention can have varying lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0074] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, also known as (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, also known as (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, also known as (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, also known as (GGS)5 (SEQ ID NO: 13).

[0075] In some embodiments, the anti-CD3ε dsFv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε dsFv antibody fragment comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0076] In some embodiments, the second antigen binding domain and the anti-CD3 binding domain are operably linked via an amino acid linker. In some embodiments, the linker within such components is primarily composed of the amino acids glycine and serine, denoted herein as a GS-linker. The GS-linker of the fusion protein of the present invention can have varying lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0077] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of: GGSGGS, also known as (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, also known as (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, also known as (GGS)4 (SEQ ID NO: 12); and GGSGGSGGSGGSGGS, also known as (GGS)5 (SEQ ID NO: 13).

[0078] In some embodiments, the cleavable linker is a polypeptide. In some embodiments, the cleavable linker is a polypeptide that is a substrate for a protease. In some embodiments, the protease is produced by a tumor near cells expressing CD3ε and / or by a tumor that co-localizes with cells expressing CD3ε in a tissue, and wherein upon exposure of the multispecific polypeptide construct to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is produced by a tumor near cells expressing one or more tumor-associated antigens (TAAs) and / or by a tumor that co-localizes with cells expressing a target TAA in a tissue, and wherein upon exposure of the multispecific polypeptide construct to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct.

[0079] In some embodiments, the cleavable linker is a polypeptide with a length of up to 50 amino acids. In some embodiments, the cleavable linker is a polypeptide with a length of up to 25 amino acids. In some embodiments, the cleavable linker is a polypeptide with a length of up to 15 amino acids. In some embodiments, the cleavable linker is a substrate for a protease selected from the proteases described herein. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, asparagine endopeptidase, interstitial protease (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP=12, MMP=13, MMP-14, and any combination thereof. In some embodiments, the cleavable linker is a substrate for a protease selected from the group consisting of uPA, asparagine endopeptidase, and interstitial protease. In some embodiments, the cleavable linker is a substrate for a matrix metalloproteinase (MMP).

[0080] In some embodiments, the multispecific construct also includes an agent coupled to the multispecific construct. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the agent is coupled to the multispecific construct via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0081] In some embodiments, the anti-multispecific constructs described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include current medical and / or surgical therapies for the intended application (e.g., cancer). For example, the multispecific constructs can be used in conjunction with additional chemotherapeutic agents or anti-tumor agents.

[0082] In some embodiments, the multispecific construct and additional agents are formulated into a single therapeutic composition, and the multispecific construct and additional agents are administered simultaneously. In some embodiments, the multispecific construct and additional agents are separated from each other, for example, each formulated into a separate therapeutic composition, and the multispecific construct and additional agents are administered simultaneously, or the multispecific construct and additional agents are administered at different times during a treatment regimen. For example, the multispecific construct is administered before the additional agents are administered, the multispecific construct is administered after the additional agents are administered, or the multispecific construct and additional agents are administered in alternating fashion. As described herein, the multispecific construct and additional agents are administered in a single dose or in multiple doses.

[0083] In some embodiments, the multispecific construct naturally contains one or more disulfide bonds. In some embodiments, the multispecific construct can be engineered to include one or more disulfide bonds.

[0084] The present disclosure also provides isolated nucleic acid molecules or polynucleotides encoding at least a portion of a multispecific construct described herein and / or one or more nucleic acid molecules encoding a multispecific construct described herein, e.g., at least a first nucleic acid encoding at least a portion of a first component of a multispecific construct and a second nucleic acid encoding at least a portion of a second component of a multispecific construct, and vectors comprising such isolated nucleic acid sequences.

[0085] Provided embodiments are polynucleotides encoding any one of the provided multispecific polypeptide constructs. Also provided are polynucleotides encoding any one of the polypeptide chains of the provided multispecific polypeptide constructs. Further provided are polynucleotides comprising a first nucleic acid sequence encoding any one of the provided multispecific constructs and a second nucleic acid sequence encoding the second polypeptide of the multispecific construct, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the multispecific polypeptide construct comprises a third polypeptide chain, and the polynucleotide further comprises a third nucleic acid encoding the third polypeptide of the multispecific construct. In some embodiments, the third nucleic acid is separated from the first and / or second polypeptide by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first and / or second nucleic acid sequence. In some embodiments, the nucleic acid encoding the self-cleaving peptide or the peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A (SEQ ID NOs: 159-164, or encoded by the sequence shown in SEQ ID NO: 165).

[0086] Provided herein are vectors comprising any of the provided polynucleotides. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector or a eukaryotic vector, optionally wherein the eukaryotic vector is a mammalian vector.

[0087] Cells comprising any of the provided polynucleotides or vectors are provided. In some cases, the cells are recombinant or isolated. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are HEK293 or CHO cells.

[0088] The present disclosure provides methods for producing a multispecific construct by culturing cells under conditions that result in expression of the multispecific construct, wherein the cells comprise the nucleic acid molecule(s). In some embodiments, the cells comprise the vector.

[0089] Provided herein are methods for producing a multispecific polypeptide construct, comprising introducing any of the provided polynucleotides or vectors into a cell and culturing the cell under conditions that result in expression of the multispecific construct to produce the multispecific polypeptide construct. Also provided are methods for producing a multispecific polypeptide construct, comprising culturing any of the provided cells under conditions where the cell expresses or produces the multispecific polypeptide. In some cases, the cell is a mammalian cell. In some embodiments, the cell is a HEK293 or CHO cell. In some embodiments, the method further comprises isolating or purifying the multispecific polypeptide construct from the cell. In some cases, the multispecific polypeptide construct is a heterodimer.

[0090] Provided herein are multispecific polypeptide constructs produced by any of the provided methods.

[0091] Provided herein are methods for stimulating or inducing an immune response, the methods comprising contacting a target cell and a T cell with any of the multispecific polypeptide constructs or pharmaceutical compositions provided herein, wherein the target cell expresses a tumor-associated antigen recognized by the multispecific polypeptide construct. In some embodiments, the target cell is a tumor cell expressing a tumor-associated antigen (TAA).

[0092] In some embodiments, the multispecific polypeptide construct comprises a cleavable linker that serves as a substrate for a protease, and the induction or stimulation of an immune response is enhanced in the presence of the protease. In some cases, the protease is produced by immune effector cells, by a tumor, or by cells present in the tumor microenvironment.

[0093] In some embodiments, the protease is produced by immune effector cells, and the immune effector cells are activated T cells, natural killer (NK) cells or NK T cells. In some cases, the immune effector cells are close to cells expressing antigens. In some embodiments, the protease is produced by tumors near cells expressing TAAs in tissues and / or is produced by tumors co-localized with TAAs in tissues, and wherein when the multispecific polypeptide construct is exposed to the protease, the protease cleaves the cleavable linker in the multispecific polypeptide construct. In some embodiments, the protease is selected from interstitial proteases, matrix metalloproteinases (MMPs), granzyme B and combinations thereof. In some cases, the protease is granzyme B.

[0094] In some embodiments, the contacting is performed ex vivo or in vitro. In some embodiments, the contacting is performed in vivo in a subject.

[0095] Provided are methods for stimulating or inducing an immune response in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some cases, the methods increase cell-mediated immunity. In some embodiments, the methods increase T cell activity. In some embodiments, the methods increase cytolytic T cell (CTL) activity. In some embodiments, the methods increase an immune response against a tumor or cancer. In some embodiments, the methods treat a disease or condition in a subject.

[0096] The present invention also provides methods for treating, preventing, or otherwise ameliorating the symptoms of one or more pathologies, delaying their progression, or alleviating symptoms associated with such pathologies by administering a multispecific polypeptide construct of the present disclosure to a subject desiring such treatment or prevention. Provided herein are methods for treating a disease or condition in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the disease or condition is a tumor or cancer.

[0097] In some embodiments of any of the provided methods, the subject (e.g., the subject to be treated) is, for example, a human or other mammal. In some embodiments of any of the provided methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), a farm animal, a draft animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0098] The multispecific polypeptide constructs of the present disclosure used in any of the embodiments of such methods and uses can be administered at any stage of the disease. For example, the multispecific polypeptide constructs can be administered to patients with cancer at any stage (from early stage to metastasis). The terms subject and patient are used interchangeably herein.

[0099] The multispecific polypeptide constructs of the present disclosure used in any of such embodiments of methods and uses can be used in a treatment regimen that includes a lead therapy.

[0100] The multispecific polypeptide constructs of the present disclosure used in any of the embodiments of such methods and uses can be administered alone or in combination with one or more additional agents, such additional agents including small molecule inhibitors, other antibody-based therapies, polypeptide- or peptide-based therapies, nucleic acid-based therapies, and / or other biologics. In some embodiments, the multispecific polypeptide constructs are administered in combination with one or more additional agents, such as, by way of non-limiting example, chemotherapeutic agents, such as alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and any other nucleic acid disrupting agents. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., ). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, such as sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, such as another antibody, such as a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteosome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulator, such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered as a standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art. In some embodiments, the multispecific polypeptide construct and the additional agent are formulated in a single composition. In some embodiments, the multispecific polypeptide construct and the additional agent are administered as two or more separate compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously. In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially.

[0101] In some embodiments, the additional agent is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0102] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an agent targeting inhibitor in the tumor microenvironment and / or a T cell or NK agonist. In some embodiments, the additional agent is radiotherapy, which is a separate combination with another additional agent (e.g., a chemotherapeutic agent or an anti-tumor agent). In some embodiments, the additional agent is a vaccine, an oncogenic virus and / or a DC activator (e.g., a toll-like receptor (TLR) agonist and / or α-CD40, as defined in non-limiting embodiments). In some embodiments, the additional agent is a tumor-targeting antibody designed to kill tumors via ADCC or via direct binding to a toxin (e.g., an antibody drug conjugate (ADC)).

[0103] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of: CTLA-4, LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of: B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is carfilzomib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of: IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, myeloid-derived suppressor cells, or T-regulatory cells. In some embodiments, the agonist is selected from the group consisting of: OX40, GITR, CD137, CD28, ICOS, CD27, and HVEM. In some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or a combination thereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, such as Yervoy TM In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, such as Opdivo TM and / or Keytruda TM .

[0104] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PDL1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is myeloid-derived suppressor cells. In some embodiments, the inhibitor is T-regulatory cells.

[0105] In some embodiments, the agonist is OX40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is CD28. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

[0106] In some embodiments, the multispecific polypeptide construct is administered during and / or after combination therapy with one or more additional agents (e.g., chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants). In some embodiments, the multispecific polypeptide construct and the additional agents are formulated into a single therapeutic composition, and the multispecific polypeptide construct and the additional agents are administered simultaneously. Alternatively, the multispecific polypeptide construct and the additional agents are separated from each other, for example, each formulated into a separate therapeutic composition, and the multispecific polypeptide construct and the additional agents are administered simultaneously, or the multispecific polypeptide construct and the additional agents are administered at different times during the treatment regimen. For example, the multispecific polypeptide construct is administered before the additional agents are administered, the multispecific polypeptide construct is administered after the additional agents are administered, or the multispecific polypeptide construct and the additional agents are administered in an alternating manner. As described herein, the multispecific polypeptide construct and the additional agents are administered in a single dose or in multiple doses.

[0107] In some embodiments, the multispecific polypeptide construct and the additional agent are administered simultaneously. For example, the multispecific polypeptide construct and the additional agent can be formulated in a single composition or administered as two or more compositions. In some embodiments, the multispecific polypeptide construct and the additional agent are administered sequentially or at different times during a treatment regimen.

[0108] In addition to the elements described above, multispecific polypeptide constructs may also contain additional elements, such as amino acid sequences at the N- or C-termini of the multispecific polypeptide construct. For example, a multispecific polypeptide construct may include a targeting moiety to facilitate delivery to a cell or tissue of interest. A multispecific polypeptide construct may be coupled to an agent such as a therapeutic agent, a detectable moiety, or a diagnostic agent. Examples of agents are shown herein.

[0109] Multispecific polypeptide constructs can also include any of the coupling reagents, linkers, and other components described herein along with the multispecific polypeptide constructs of the present disclosure.

[0110] The present disclosure also relates to immunoconjugates comprising a multispecific polypeptide construct conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioactive isotope (i.e., a radioconjugate). Cytotoxic agents suitable for targeting diseased T cells in, for example, T-cell derived lymphomas include, for example, dolestatin and its derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). In some embodiments, the agent is dolestatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0111] In some embodiments, the linker between the multispecific polypeptide construct and the cytotoxic agent is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, two or more linkers are present. The two or more linkers can be identical, e.g., both cleavable and non-cleavable, or the two or more linkers can be different, e.g., at least one cleavable and at least one non-cleavable.

[0112] Multispecific polypeptide constructs and conjugates thereof can be used in methods for treating a variety of conditions and / or diseases. Non-limiting examples of diseases include: all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, and pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, ischemia, etc. For example, indications would include leukemia (including T-cell acute lymphoblastic leukemia (T-ALL)), lymphoblastic diseases (including multiple myeloma), and solid tumors (including lung tumors, colorectal tumors, prostate tumors, pancreatic tumors, and breast tumors (including triple-negative breast cancer)). For example, indications include bone disease or cancer metastasis that is unrelated to the source of the primary tumor; breast cancer, including (by way of non-limiting embodiment) ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as, by way of non-limiting embodiment, non-small cell lung cancer; multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as, by way of non-limiting embodiment, renal cell carcinoma; and / or skin cancer, such as, by way of non-limiting embodiment, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0113] Also provided are pharmaceutical compositions comprising any of the multispecific polypeptide constructs provided herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition is sterile. The pharmaceutical compositions of the present disclosure may include a multispecific polypeptide construct of the present disclosure and a carrier. Such pharmaceutical compositions may be included in a kit (e.g., a diagnostic kit).

[0114] Those skilled in the art will appreciate that the antibodies disclosed herein have a variety of uses. For example, the proteins disclosed herein are used as therapeutic agents for a variety of conditions. The antibodies disclosed herein are also useful as reagents in diagnostic kits or as diagnostic tools, or such antibodies can be used in competitive assays to generate therapeutic agents.

[0115] Brief description of the attached figure

[0116] Figure 1 Schematic diagram of the essential components of a multispecific polypeptide construct of the present invention with restricted CD3 binding. The antigen-binding domain is located at the amino and / or carboxyl termini. The Fc region (e.g., a heterodimeric Fc region) is located N-terminal to the CD3-binding domain. This positioning of the Fc region in close proximity to the CD3-binding domain hinders CD3 binding.

[0117] Figure 2 is a diagrammatic illustration showing exemplary structures of multispecific molecules of the present disclosure containing a cleavable linker and having dual effector functions, wherein proteolytic cleavage of the cleavable linker results in activation of the multispecific polypeptide construct to produce two components, each with biological activity.

[0118] Figure 3 Schematic diagram of various FRα-targeting restricted CD3 constructs composed of two polypeptides (i.e., chain 1 and chain 2). The above figure provides an exemplary display of a cleavable multispecific polypeptide construct containing a cleavable linker with one or more protease substrate recognition sites for, for example, MTSP1, MMP, and / or granzyme B. Chain 1 contains a FRα sdAb (antigen binding domain) linked to a heterodimeric Fc "hole," to an anti-CD3 VL domain via a protease cleavable linker (cx1547: granzyme B only, cx309: MTSP1, MMP, and granzyme B), and to a second FRα sdAb. Chain 2 contains a FRα sdAb linked to a complementary heterodimeric Fc "knob," to an anti-CD3 VH domain via the same protease linker as described above, and to a second FRα sdAb. The lower panel of Figure 6 shows a similar configuration to the upper panel, except that the linker is a non-cleavable linker (ranging from 3 amino acids in cx 1356 to 18 amino acids in cx681). Upon co-expression, the CD3 binding domains assemble properly via VL:VH association on the hole and knob, respectively.

[0119] Figures 4A-4C Constructs generated to compare the effect of linkers on limiting CD3 binding in the generated constructs are shown. Figure 4A An embodiment of a multispecific polypeptide construct containing the same cleavable linker in each polypeptide chain to couple each Fc polypeptide of the heterodimeric Fc to a domain of the CD3 binding region is shown (exemplary construct cx 1762 is shown). Figure 4A In the format shown in , the construct is shown in an undecomposed state. Figure 4B An alternative form of construct is shown in , in which only a single cleavable linker is employed to connect the Fc region to the CD3 binding domain, designated hemi-cleaved (exemplary construct cx3238 is shown). Figure 4C Display Representative Figure 4A and 4B Figure 2 shows the structure of the constructs in their fully cleaved form (exemplary construct cx2190 is shown). Constructs representing proteolytic cleavage products can be recombinantly generated by co-expressing the various chains shown. The FRα-targeting sdAb is located at the C-terminal position of each construct.

[0120] Figures 5A-5ERepresentative EGFR-targeted and EGFR / cMET dual-targeted restricted CD3 engagers are shown. Figure 5A In , cx2513 has an EGFR-targeted sdAb located at the C-terminus of each chain of the heterodimer and thus exhibits bivalent binding to EGFR. Figure 5B In the present invention, cx3030 has EGFR-targeted sdAbs located at the N- and C-termini of each chain of the heterodimer and thus exhibits tetravalent binding to EGFR. Figure 5C In , cx2973 has a cMET-targeted sdAb located at the N-terminus and an EGFR-targeted sdAb located at the C-terminus of each chain of the heterodimer and thus exhibits bivalent binding to each of cMET and EGFR. Figure 5D In the present invention, cx2979 has a cMET-targeted sdAb located at the N-terminus of one chain of the heterodimer and an EGFR-targeted sdAb located at the C-terminus and thus exhibits monovalent binding to cMET and bivalent binding to EGFR. Figure 5E In the present invention, cx2977 has a cMET-targeted sdAb located at the N-terminus of one or the other chain of the heterodimer and an EGFR-targeted sdAb located at the C-terminus and thereby exhibits monovalent binding to each of cMET and EGFR.

[0121] Figure 6A and 6B Schematic diagram of the component chains used to assemble an exemplary B7H3-targeted restricted CD3 binder. The B7H3 binding domains utilized in such representative constructs include sdAbs, scFvs, or FABs. Typically, constructs containing sdAbs and scFvs consist of two heterodimeric chains, while constructs containing FABs include a third chain of the cognate light chain (VL-CL).

[0122] Figure 7 5T4-targeted restricted CD3 binders are shown. The core of the generated molecule contains a heterodimeric Fc followed by a cleavable linker and a disulfide-stabilized anti-CD3 Fv. The TAA binding portion of such molecules is placed at the N- or C-terminus of the heterodimeric Fc chain. In the top column, the TAA binding unit is a Fab consisting of a Fd (VH-CH1) at the N-terminus of the knob polypeptide and a C-terminus of the hole polypeptide. In cases where the Fab is the binding unit, a third chain (light chain - VL-CL) is expressed to associate with the Fd. In the middle and bottom columns, the TAA binding units are single domain antibodies with the knob polypeptide located at the N- and C-termini. In the middle column, the TAA binding sdAbs of the generated constructs are identical, while in the bottom column, the TAA binding sdAbs of the generated constructs are different sequences with different epitopes.

[0123] Figure 8Schematic diagram of a representative CD20-targeted constrained CD3 engagement construct, cx3309, in which the CD20 binding domain is a scFv derived from the CD20 antibody GA101.

[0124] Figure 9 Schematic diagram of a representative DLL3-targeted, restricted CD3-engaging construct, cx3308, in which the DLL3-binding domain is a scFv. This exemplary construct comprises two chains, each with complementary components of a heterodimeric Fc linked to a CD3-binding domain and one component of a DLL3-binding scFv. In assembled form, the construct is bivalent for DLL3 and has the CD3-binding domain located at the C-terminus of the Fc heterodimer.

[0125] Figure 10A The images are SDS-PAGE images of a representative FRα targeting restricted CD3 engagement construct cx 1547 under reducing (R) and non-reducing (NR) conditions. The expected molecular weight is 135 kDa. Figure 10B and Figure 10C is a picture of a chromatogram from size exclusion analysis of cx 1547, demonstrating that it is a single species with a measured molecular weight of 137.9 kDa. Figure 10C yes Figure 10B A magnified view near the main peak is shown in .

[0126] Figure 11A and 11B is a pair of graphs demonstrating the ability of an exemplary multispecific polypeptide construct of the present disclosure (referred to herein as cx309) to bind to human T cells in either uncleaved or proteolytically cleaved states. Figure 11A and Figure 11B cleavage of cx309 in .

[0127] Figures 12A-12D Cellular binding by representative FRα targeting restricted CD3 engagement constructs cx1356, cx681 and cx1547 is shown. Figure 12A and Figure 12C Shown to bind to Ovcar5 cells (FRα-positive ovarian cancer cell line). Figure 12B and Figure 12D Shows no binding to T cells. Figure 12A and Figure 12B Histograms showing normalized cell counts versus fluorescence at 100 nM for each construct are shown. Complete titrations of each construct on various cell types are shown in Figure 12C and Figure 12D In. Figure 12A and Figure 12BIn the Figures, the secondary anti-human APC antibody only control is shown in the filled black trace, while the positive control anti-CD3 binding is shown in the open trace, and cx1356, cx681, and cx1547 are shown in the grey shaded trace.

[0128] Figures 13A-13B Cellular binding by a representative EFGR targeting restricted CD3 engagement construct cx3030 is shown. Figure 13A Binding is shown to the EGFR-positive cell line Colo-205 at 100 nM. Figure 13B No binding to T cells at 100 nM is shown. Binding is shown as a histogram of normalized cell counts versus fluorescence. The secondary anti-human APC antibody control alone is shown in the filled black trace, while positive control anti-CD3 binding is shown in the open trace, and cx3030 is shown in the gray shaded trace.

[0129] Figures 14A-14D B7H3 targeting restricted CD3 adaptor binding to B7H3 positive A375 ( Figure 14A and 14B ) and unbound to CD3 on T cells ( Figure 14C and 14D ). The alternative format DART-Fc targeting B7H3 and CD3 exhibits strong binding to B7H3 and CD3 on T cells. Various B7H3 antigen binding domains were used herein, including cx3095sdAb, cx3313FAB, and cx3314scFv. The scFv and FAB contain the same anti-B7H3 VH and VL sequences used in the DART-Fc format. Figure 14A and 14C Comparative histograms are shown for each construct at a concentration of 100 nM. A secondary anti-human APC antibody only control is shown in the filled black trace, and various B7H3-targeted CD3-engaging constructs are shown in the white non-shared traces. Figure 14B and 14D Titrations of binding by various constructs to BH73 and CD3, respectively, are shown.

[0130] Figures 15A-15B Cellular binding by representative 5T4 targeting-restricted CD3 engagement constructs cx3262 and cx3315 is shown. Figure 15A Binding to 5T4 positive cell line Ovcar-5 at 400 nM is shown. Figure 15B No binding to T cells at 400 nM is shown. Binding is shown as a histogram of normalized cell counts versus fluorescence. The secondary anti-human APC antibody control alone is shown in the filled black trace, while positive control anti-CD3 binding is shown in the open trace, and cx3262 and cx3315 are shown in the gray shaded trace.

[0131] Figures 16A-16D Cellular binding by the representative CD20 targeting restricted CD3 engagement construct cx3309 is shown. Figure 16A and Figure 16C Binding is shown to Ramos cells, a CD20-positive cell line. Figure 16B and Figure 16D Shows no binding to T cells. Figures 16A-16B Histograms showing normalized cell counts versus fluorescence at 100 nM for each construct are shown. Complete titrations of each construct on various cell types are shown in Figures 16C-16D Secondary anti-human APC antibody only control is shown in the filled black trace, while positive control anti-CD3 binding is shown in the open trace, and cx3309 is shown in the grey shaded trace.

[0132] Figure 17 is a graph showing the ability of lysed or uncleaved cx309 to activate the CD3 NFAT reporter Jurkat cell line (Promega, USA) in the presence or absence of FRα-expressing cellular Ovcar5.

[0133] Figure 18 Antigen-dependent T cell activation by cx1547 is shown. Various cell lines that are FRα-positive (T47D, IGROV1, NCI-H2342, Ovcar-5, Skov-3, and A2780) or negative (NCI-H460) were co-incubated with the Jurkat CD3 NFAT-GFP reporter cell line and fluorescence was measured at 6 hours. This demonstrates the ability of the restricted CD3 construct to activate T cells in an antigenic manner.

[0134] Figures 19A-19D It was shown that if proteolysis occurs within the linker between the Fc domain and the CD3 domain, the T cell activation capacity of the restricted CD3 binder is enhanced. Figure 19A ) or FRα-negative CCRF-CEM cells ( Figure 19B ) in the presence of 20 nM cx 1762, cx3238 or cx2190 mediated T cell activation kinetics. Also shown here is the activation of FRα positive Ovcar-5 cells ( Figure 19C ) or FRα-negative CCRF-CEM cells ( Figure 19DThe efficacy of T cell activation mediated by cx1762, cx3238, or cx2190 in the presence of α-glucose ...

[0135] Figures 20A-20D is a series of graphs demonstrating the antigen-dependent T cell activation capacity of various EGFR and EGFR / cMET targeting-restricted CD3 engagers. Notably, T cell activation capacity is enhanced with increasing valency or additional target antigen binding specificity. The kinetics of T cell activation mediated by various constructs on antigen-positive A431 cells are shown in Figure 20A The kinetics of T cell activation mediated by various constructs on antigen-negative CCRF-CEM cells are shown in Figure 20C The T cell activation efficacy of various constructs on antigen-positive A431 cells was shown in Figure 20B The T cell activation efficacy of various constructs was shown in Figure 20D Here, the Jurkat CD3NFAT-GFP reporter cell line was used.

[0136] Figures 21A-21B The ability of a representative B7H3 targeting restricted CD3 engagement construct, cx3095, and alternative DART-Fc formats targeting both B7H3 and CD3 to mediate target antigen-specific T cell activation is shown. Jurkat CD3 NFAT-GFP reporter cells were used to evaluate the activation of target antigen-specific T cells in the B7H3-positive cell line A375 ( Figure 21A ) and the B7H3 negative cell line Raji ( Figure 21B ) in the presence of T cells.

[0137] Figures 22A-22F The ability of representative B7H3-targeted restricted CD3 engagement constructs and alternative DART-Fc formats targeting B7H3 and CD3 to mediate target antigen-specific T cell activation is shown. Notably, the restricted CD3 engagement constructs utilize sdAbs, scFvs, or FABs targeting B7H3. Jurkat CD3 NFAT-GFP reporter cells were used to evaluate the effect of B7H3 on the B7H3-positive cell line A375 ( Figure 22A 、 22C , 22E) and the B7H3-negative cell line CCRF ( Figure 22B 、 22D, 22F) in the presence of T cells. Figure 22A and 22B ) or 2 nM ( Figure 22C The kinetics of T cell activation mediated by each construct of 22D or 22D are also shown. Figure 22E ) and negative ( Figure 22F ) cell lines.

[0138] Figures 23A-23B Figure 1 is a series of graphs showing the T cell activation capacity of 5T4 targeting restricted CD3 engager constructs. This embodiment shows how bivalent bi-epitope TAA targeting increases the activity of restricted CD3 engagers relative to bivalent mono-epitope proteins on TAA-positive cells (OVCAR5). In the presence of TAA-negative cells (CCRF), none of the constructs induce T cell activation.

[0139] Figure 24 Figure 2 is a graph showing the ability of a representative 5T4 targeting-restricted CD3 engagement construct, cx3315, to induce antigen-dependent T cell activation. T cell activation by cx3315 in the presence of a 5T4-positive cell line (OVCAR5) and a 5T4-negative cell line (CCRF-CEM) was monitored using a Jurkat CD3 NFAT-GFP reporter cell line.

[0140] Figure 25 Figure 3. The ability of a representative CD20-targeting restricted CD3-engaging construct, cx3309, to induce antigen-dependent T cell activation. T cell activation by cx3309 in the presence of the CD20-positive Ramos cell line and the CD20-negative CCRF-CEM cell line was monitored using the Jurkat CD3 NFAT-GFP reporter cell line.

[0141] Figure 26 Figure 2 is a graph showing the ability of a representative DLL3 targeting-restricted CD3-engaging construct, cx3308, to induce T cell activation. T cell activation by cx3309 in the presence of SHP-77 cells (which are DLL3 positive) was monitored using the Jurkat CD3 NFAT-GFP reporter cell line. This demonstrates that scFv moieties can be used to target TAAs in a CD3-restricted format and effectively activate T cells when bound to cognate TAA-positive cell lines.

[0142] Figures 27A-27F The effect of linker length on the expression of IGROV1 in FRα-positive cells was shown. Figure 27A 、 27C , 27E) or FRα-negative NCI-H460 ( Figure 27B 、 27D, 27F) in the presence of the ability to activate T cells. Figures 27A-27B Shown are the kinetics of T cell activation by 2 nM of the various constructs on antigen-positive and -negative cells, respectively. Figures 27C-27D Shown are the magnitudes of T cell activation capacity by 2 nM of each construct on antigen-positive and -negative cells, respectively. Figures 27E-27F The efficacy of various constructs with different linker lengths on antigen-positive and antigen-negative cells is shown. T cell activation was evaluated using the Jurkat CD3NFAT-GFP reporter cell line. When bound to a second antigen on the target cell, the restricted CD3 protein effectively engages and clusters CD3 only on T cells.

[0143] Figures 28A-28C Demonstrates antigen-dependent T cell-mediated cytotoxicity by CX1547. Figure 28A CX1547 was shown not to induce T cell-mediated cytotoxicity in an antigen-negative cell line (NCI-H460). Figure 28B CX1547 was shown to induce T cell-mediated cytotoxicity against an antigen-positive cell line (Ovcar5). Figure 28C Shown are the kinetics of T cell-mediated cytotoxicity against OVCAR5 cells induced by 3 nM cx1547. cx1547 induces T cell-mediated cytotoxicity only in antigen-positive cell lines. Cytotoxicity was monitored on an Incucyte ZOOM Imager using a caspase-3 / 7 fluorescent probe substrate on differentially labeled target cells. Effector to target cell ratios (E:T) of 20:1 and 10:1 were evaluated in this assay.

[0144] Figures 29A-29F The kinetics of T cell-mediated cytotoxicity driven by representative B7H3 targeting-restricted CD3-engaging constructs and alternative DART-Fc formats targeting both B7H3 and CD3 are shown. The titration range of 50 nM to 80 pM of the CD3-engaging constructs on the B7H3-positive A375 cell line is shown. Figures 29A-29E middle. Figure 29F Measurements of 50 nM of each construct on A549 cells in which B7H3 expression had been knocked down are shown. Notably, all constructs displayed B7H3-dependent T cell-mediated cytotoxicity.

[0145] Figure 30 Shown are the magnitudes of T cell-mediated cytotoxicity induced by 2.5 nM B7H3 targeting restricted CD3 engagement constructs and DART-Fc B7H3 x CD3 formats on antigen-positive (A375) and negative (A549-B7H3 knockdown) cell lines.

[0146] Figures 31A-31FTwo forms of FRα-targeted CD3 binders were shown to induce FRα-positive Ovcar-5 cells ( Figures 31A-31E ) and FRα-negative NCI-H60 cells ( Figure 31F )-mediated T cell cytotoxicity. CX2190 is a representative C-terminal product that can be derived from the proteolytic processing of granzyme B by CX1762. Notably, CX2190 exhibited superior efficacy compared to CX1792, demonstrating a significant enhancement of CD3 binding mediated by proteolysis within the linker region between the Fc and CD3 binding domains. The kinetics of T cell-mediated cytotoxicity on FRa-positive cells at 20 nM, 32 pM, and 6 pM, respectively, are shown in Figure 2. Figure 31A 、 31B and 31C. Figure 31D and Figure 31E The efficacy of the two forms of the FRa CD3 engager is shown at 24 and 40 hours, respectively. Panel F demonstrates that in the absence of FRa expression on target cells, no significant cytotoxicity was mediated by any of the constructs.

[0147] Figure 32 Figure 3. T cell-mediated cytotoxicity mediated by a representative 5T4-targeted, CD3-restricted construct, cx3315. CX3315 induces specific T cell cytotoxicity against the 5T4-expressing cell line, Ovcar-5, but not against the 5T4-negative cell line, CCRF-CEM. 20 nM CX3315 was used in this analysis.

[0148] Figure 33 This graph shows T cell activation after 20 hours of co-culture of T cells with Ovcar5 cells in the presence or absence of cleaved or uncleaved cx309. Only cleaved cx309 mediates FRα-dependent T cell activation via CD3 binding. T cell activation was monitored by flow cytometric analysis of CD25% CD4 and CD8 populations.

[0149] Figures 34A-34H Activation of CD4 by representative B7H3 targeting-restricted CD3 engagement constructs and alternative DART-Fc formats targeting B7H3 and CD3 was shown in a target-dependent manner ( Figure 34A and 34E ) and CD8( Figure 34C and 34G ) T cells. T cells were isolated from the B7H3 positive cell line A375 ( Figure 34A 、 34C , 34E, 34G) or B7H3 knockdown A549 cell lines ( Figure 34B 、 34D , 34F, 34H) and were incubated with the activation markers CD25 and CD71 by flow cytometry. These data demonstrate the B7H3-dependent T cell activation capacity of the constructs used.

[0150] Figure 35 The ability of the B7H3-targeted, restricted CD3-engaging construct cx3095 to mediate antigen-dependent INFγ production was demonstrated. Cytokine production was quantified using an INFγ ELISA. A375 was used as a B7H3-positive cell line, while the B7H3 knockdown A549 cell line was used as a negative cell line.

[0151] Figures 36A-36B A representative FRα-targeted restricted CD3-engaging construct, cx1547, is shown to induce FRα-dependent IFNγ from human PBMCs. Figure 36A ) and IL-2( Figure 36B Cytokine production was measured using FluoroSpot cytokine capture assays. IGROV-1 and NCI-H460 were used as FRα-positive and negative cell lines, respectively.

[0152] Figure 37 The ability of the B7H3-targeted, restricted CD3-engaging construct cx3095 to mediate antigen-dependent INFγ production was demonstrated. Cytokine production was monitored using FluoroSpot analysis. A375 and CCRF-CEM cell lines were used as B7H3-positive and -negative cell lines, respectively.

[0153] Figures 38A-38D The ability of the FRα-targeting restricted CD3 construct cx1547 to activate T cells present in dissociated primary human ovarian tumor samples and to elicit cytotoxicity was demonstrated. Figure 38A Flowchart showing the relative prevalence of tumor cells (EpCAM+) and infiltrating lymphocytes (CD45+) in dissociated ovarian tumor samples. Figure 38B Shown are the viability of adherent tumor cells after 6 days of incubation following treatment with a common FRα antibody or cx1547 (CellTiterGlo). Figure 38C Shown is INFγ production after 6 days of incubation following treatment with FRa antibody or cx1547. Figure 38D Representative images showing remaining adherent tumor cells after 6 days of treatment with no antibody (left panel), a common FRα antibody (center panel), or cx1547 (right panel). Implementation Method

[0154] The present invention provides a restricted T cell engagement fusion protein in the form of a multispecific polypeptide construct that binds at least CD3 and a second antigen. The multispecific polypeptide construct provided herein includes at least a first component, which includes one or more copies of an antigen binding domain that binds an antigen that is operably linked to the Fc region of an immunoglobulin; a second component, which includes one or more copies of at least one binding domain that binds to CD3 (referred to herein as an anti-CD3 binding domain or CD3 binding region, which are terms used interchangeably herein); and a linker, such as a polypeptide linker, that connects the first component to the second component. In some embodiments, the antigen is a tumor-associated antigen (TAA). In some embodiments, the linker is a cleavable linker.

[0155] The multispecific polypeptide constructs provided include configurations in which a first component containing an Fc region is at the N-terminus of a second component containing a CD3 binding region. In this embodiment, the first and second components are connected via a linker at the C-terminus of the Fc region. In some embodiments, the antigen binding domain is located on the amino terminal (N-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen binding domain is located on the carboxyl terminal (C-terminal) region of the multispecific polypeptide construct. In some embodiments, the antigen binding domain is located on the N- and C-terminal regions of the multispecific polypeptide construct. Various configurations of multispecific polypeptide constructs as provided herein are shown in Figure 1 middle.

[0156] The multispecific polypeptide constructs provided exhibit limited T cell engagement activity because once the antigen is bound via the antigen binding domain, such constructs are only substantially bound to CD3. This is exemplified in the embodiments and figures provided herein, which show that limited CD3 engaging proteins effectively bind to TAA-positive cells while rarely binding to non-binding T cells. This unique property allows limited CD3 engaging proteins to be distributed to sites where TAAs are present without binding to peripheral T cells. This form is different from other CD3 engaging multispecific constructs in that constitutive CD3 binding is not allowed or eliminated, thereby providing significant benefits that avoid peripheral T cell binding and allowing preferential distribution to sites of antigens such as those recognized by the antigen binding domain. For example, as shown in the embodiments, the limited CD3 engaging form can have similar efficacy to the DART-Fc form (e.g., disclosed PCT invention No. WO2017 / 030926), but the binding to peripheral T cells is significantly weakened. In addition, other CD3 engaging constructs mediate antigen-dependent T cell activation, however, the multispecific polypeptide constructs provided herein mediate antigen-dependent T cell binding and activation.

[0157] In some aspects, the limited T cell engagement activity of the provided multispecific polypeptide construct is due to the Fc region being positioned at the N-terminus of the CD3 binding region. In some embodiments, this positioning reduces, weakens, suppresses and / or prevents CD3 binding by the CD3 binding region. In the absence of antigen binding by the antigen binding domain, the multispecific polypeptide construct provided herein exhibits reduced or eliminated CD3 binding and T cell activation capabilities. In some embodiments, in the presence of an antigen binding event mediated by the antigen binding domain of the multispecific polypeptide construct, the ability of the CD3 binding region to bind CD3 is greatly enhanced. In some embodiments, in the presence of an antigen binding event mediated by the antigen binding domain of the multispecific polypeptide construct, the ability to activate T cells is greatly enhanced. Engagement of its cognate antigen by the antigen binding domain within the multispecific polypeptide construct results in subsequent T cell engagement and mediating antigen-dependent T cell activation, such as cytotoxicity, cytokine release, degranulation and proliferation. In some embodiments, the provided multispecific polypeptide construct can be used to increase an immune response, such as enhancing T cell activity, including cytolytic (or cytotoxic) T cell activity. In some aspects, modulation of the immune response treats a disease or condition in a subject.

[0158] In some embodiments, one or more antigen binding domains bind to an antigen on a tumor cell or a cell of the tumor microenvironment. In some aspects, the provided multispecific polypeptide constructs can be used to increase an immune response against a tumor or cancer, such as T cell activity, such as cytotoxic activity. In some embodiments, the provided multispecific polypeptide constructs can be used to treat a tumor or cancer in a subject.

[0159] The multispecific polypeptide constructs of the present disclosure ensure that CD3 in peripheral blood does not bind to T cells, as the CD3 binding region of such constructs is limited or otherwise blocked and / or inhibited by the presence of the Fc region. Therefore, the multispecific polypeptide constructs of the present disclosure provide multiple advantages. In some aspects, such constructs limit the trough effect of binding to all T cells. In some aspects, such constructs reduce systemic toxicity.

[0160] In some embodiments, the multispecific polypeptide constructs provided herein allow for controlled biodistribution to desired sites in a subject, such as sites of tumor-associated antigen (TAA) expression. Sites of TAA expression include, for example, tumors and the surrounding tumor microenvironment.

[0161] In some embodiments, the multispecific polypeptide constructs of the present disclosure exhibit specificity for CD3 and one or more other antigens. In some embodiments, the multispecific polypeptide constructs may contain more than one antigen binding domain that can bind to one or more TAAs, such as 2, 3, or 4 antigen binding domains, for example, see Figure 1. In some embodiments, one or more antigen binding domains bind to the same antigen. In some embodiments, the multispecific polypeptide construct includes more than one antigen binding domain that binds to different epitopes of the same antigen. In some embodiments, the multispecific polypeptide construct includes more than one antigen binding domain that binds to one or more different antigens. In some embodiments, the multispecific polypeptide construct includes more than one antigen binding domain that binds to different epitopes on the same antigen, and includes additional antigen binding domains that bind to one or more different antigens. In some aspects, the multispecific polypeptide construct provided is a bispecific polypeptide construct, such that it can bind to CD3 and another antigen (e.g., TAA) via the binding of the antigen binding domains of the multispecific polypeptide construct. In some embodiments, the multispecific polypeptide construct provided is a bispecific polypeptide construct that provides tetravalent engagement of one or more TAAs through the use of a first antigen binding domain and a second antigen binding domain. For example, in some embodiments, the bispecific polypeptide construct includes a first antigen binding single domain antibody (sdAb) and a second antigen binding sdAb, such as Figure 1 and 2 As shown in .

[0162] In some embodiments, the multispecific polypeptide constructs provided herein exist in two states with respect to their ability to bind CD3 and subsequently activate T cells: (1) an "inactive," i.e., uncleaved, state that occurs in the absence of any binding by any or all of the antigen binding domains, such that CD3 binding is restricted and precludes T cell interactions, and (2) an "active," state that occurs upon antigen binding by any or all of the antigen binding domains, such that the CD3 binding region is able to bind CD3 and is permissive for T cell interactions.

[0163] In some embodiments, the Fc region is connected to the CD3 binding domain via one or more linkers. In some embodiments, the Fc region is connected to the CD3 binding domain via one or more non-cleavable linkers. In some embodiments, the Fc region is connected to the CD3 binding domain via a cleavable linker or an otherwise labile linker.

[0164] In some embodiments, the Fc region and the CD3 binding region are connected by a cleavable linker, wherein in some aspects, enhanced CD3 binding occurs after cleavage of the cleavable linker. In some such aspects, the "active" state can be further amplified through several mechanisms, including through cleavage of the linker connecting the CD3 binding region to the Fc region. In some embodiments, the cleavable linker is a linker that contains a substrate recognition site for a protease. In some embodiments, wherein the Fc region and the CD3 binding region are connected by a cleavable linker, enhanced CD3 binding can occur after cleavage within the linker.

[0165] In some aspects, the multispecific polypeptide constructs of the present disclosure allow for therapeutic efficacy in the absence of proteolysis.

[0166] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region is a heterodimeric Fc region. In some embodiments, the Fc region is a monomeric Fc region. In some embodiments, the Fc region of the multispecific polypeptide construct can interact with FcγR and mediate innate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the Fc region of the multispecific polypeptide construct can interact with complement proteins (i.e., C1q) and mediate complement-dependent cytotoxicity. Therefore, in some aspects, the multispecific polypeptide construct of the present disclosure allows for multiple immune effector mechanisms, including innate immune effectors and T cells.

[0167] In some embodiments, wherein the Fc region and the CD3 binding region are operably connected by a cleavable linker, cleavage of the linker between the Fc region and the CD3 binding region can separate the multispecific polypeptide construct into a first and a second component. Depending on the composition of the multispecific polypeptide construct, the first and second components may have different functionalities. In some embodiments, the Fc region is a region that exhibits one or more effector functions (e.g., ADCC, CDC, or ADCP functions). In such examples, the multispecific polypeptide constructs of the present disclosure can be used to generate self-amplification systems. For example, the multispecific construct can be used as follows: ADCC mediated by NK cells after TAA targeting and CD16 binding in the Fc region causes the release of granzyme B, which can decompose and cleave the linker extracellular protein between the first and second components of the multispecific polypeptide construct.

[0168] In some embodiments, the linker is a cleavable linker. The multispecific polypeptide construct provides a two-in-one therapeutic moiety with dual effector functions, wherein proteolytic activation of the multispecific polypeptide construct produces two components that are each biologically active. The multispecific polypeptide constructs of the present disclosure can provide only Fc-mediated effector functions, such as ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC.

[0169] It is expected that the restricted CD3 engagement construct is suitable for use with any TAA binding domain, thereby allowing for better therapeutic exposure in the tumor or tumor microenvironment by avoiding interaction with peripheral T cells and mediating potent TAA-dependent T cell cytotoxicity. The inclusion of a protease-cleavable linker between the Fc and the components of the CD3 binding domain enables amplification of T cell activation capacity by allowing the CD3 binding domain to be fully exposed. Depending on the specific linker included, the amplification step can be mediated by tumor-associated proteases or granzymes released after antigen-dependent T cell activation. If a tumor protease-cleavable linker is included, the amplification is mediated by the tumor or tumor microenvironment. If a granzyme B-cleavable linker is included, the amplification can be mediated by T cells after antigen-dependent activation. In addition, in the case where an effector-activated Fc is included in the construct, the amplification can be mediated by granzymes released from NK cells, which occurs via an ADCC mechanism.

[0170] In some embodiments, the protease is a protease produced in the tumor microenvironment and / or induced by the initial binding of the CD3 binding region to CD3 in the tumor microenvironment via the combination of the antigen binding domain and TAA when T cells are activated. In some embodiments, the protease is granzyme B. In some aspects, the multi-specific polypeptide construct of the present invention affects the ability of the protease and / or granzyme B cleavage Fc immunoglobulin polypeptide below the multi-specific polypeptide construct to produce two therapeutically active proteins with different effector cell engagement in some cases. In some aspects, when the cleavable linker is cleaved, the first part or component of the cleavage retains the Fc-effector function and the bivalent targeting of the first antigen (e.g., TAA) via the first antigen binding domain, and the second part or component retains the ability of T cell engagement because the separation of the CD3 binding region and the Fc region allows CD3 to bind. The second part or component of the cleavage also retains the ability to bind to TAA in some cases, which can be a bivalent binding via the second antigen binding domain.

[0171] In some embodiments, the second portion or component contains a CD3 binding region that is monovalent to CD3, such that T cells will not be activated unless a TAA is present. In some aspects, if the multivalent polypeptide construct contains a cleavable linker, the cleaved second portion or component allows for TAA-dependent T cell-mediated cytotoxicity. In some cases, the cleaved second portion or component ensures that there is no FcRn interaction. In addition, the size of the cleaved second portion or component will be small enough, for example, only about 50 kDa, to ensure rapid release if for any reason the cleaved second portion or component is distributed outside the tumor site after cleavage and / or if it is abnormally cleaved outside the tumor site.

[0172] In some embodiments, the multispecific polypeptide constructs of the present disclosure allow for simultaneous T cell and NK cell-mediated cytotoxicity. In some cases, this activity can occur in a multispecific polypeptide construct comprising a first antigen-binding domain (e.g., a first anti-TAA antigen-binding domain) and a second antigen-binding domain (e.g., a second anti-TAA antigen-binding domain) that can target different and / or non-competing epitopes on a given TAA.

[0173] In some aspects, the multispecific polypeptide constructs of the present disclosure provide many advantages over current bispecific therapeutics. The multispecific polypeptide constructs of the present disclosure are smaller than commonly used therapeutic antibodies, for example 150 kDa versus 125 kDa, which will allow for better target (e.g., tumor) penetration. First, the size of the entire multispecific polypeptide construct provides a long half-life for the uncleaved construct, and when the construct is cleaved, the second portion or component of the cleavage will be small enough to ensure a short half-life. In some aspects, the multispecific polypeptide constructs of the present disclosure exhibit reduced systemic toxicity or reduced toxicity to any area outside the tumor and / or tumor microenvironment because CD3 binding by the CD3 binding region depends on TAA engagement before CD3 engagement occurs. In some cases, the inclusion of a cleavable linker that is specific for proteases in the tumor environment reduces CD3 binding by the multispecific construct until proteolytic activation and TAA engagement, thereby amplifying CD3 engagement.

[0174] The multispecific polypeptide constructs disclosed herein are designed to ensure that the protease that cleaves the cleavable linker does not need to be tumor-biased (e.g., it does not need to be differentially expressed only at the tumor site and / or in the tumor environment). Instead, such multispecific polypeptide constructs only require that the protease and TAA be present at the same location. The valency of such constructs will drive biodistribution and retention in the tumor and / or tumor microenvironment.

[0175] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated as being incorporated herein by reference. The citation of publications and patent documents is not intended to be an admission that any one is relevant prior art, nor is it intended to constitute any admission as to the contents or dates of such publications and patent documents. Now that the invention has been described in writing, those skilled in the art will recognize that the invention can be practiced in various embodiments and the following description and embodiments are provided for illustrative purposes only, not to limit the claims below.

[0176] I. Definition

[0177] Unless otherwise defined, the scientific and technical terms used in connection with the present invention should have the meanings commonly understood by those skilled in the art. The term "one" entity or "a" entity refers to one or more of these entities. For example, a compound refers to one or more compounds. Therefore, the terms "one", "a kind of", "one or more" and "at least one" are used interchangeably. In addition, unless the context otherwise requires, singular terms will include plural terms and plural terms will include singular terms. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein are those known and commonly used in the industry. Standard techniques are used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are implemented according to the manufacturer's instructions, or are completed as conventional methods in the industry, or are implemented as described herein. Generally, the above-mentioned techniques and procedures are implemented according to conventional methods known in the industry and as described in various general and more specific references, and such references are cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0178] As utilized in accordance with the present invention, the following terms shall be understood to have the following meanings unless otherwise indicated:

[0179] As used herein, the term "antibody" refers to immunoglobulin molecules and antigen-binding portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not bind to other polypeptides or binds with much lower affinity (K). d >10 -6 ) binding. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, fully human, domain antibodies, single chain, Fab, and F(ab')2 fragments, Fv, scFv, and Fab expression libraries.

[0180] It is known that the basic antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Generally speaking, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG3, IgG4, and others. In addition, in humans, the light chain can be a kappa chain or a lambda chain.

[0181] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species, which antibody molecule consists of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules in the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen and are characterized by a unique binding affinity for the antigen.

[0182] The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches of sequences within the V regions of the heavy and light chains, called "hypervariable regions," are inserted between more conserved flanking stretches of sequences called "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences found naturally between and near the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." The distribution of amino acids within each domain is as defined in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia and Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

[0183] As used herein, the term "epitope" includes any specific portion of the antigen targeted by an antibody, antibody fragment or other binding domain. The term "epitope" includes any protein region to which specific binding is directed. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitope determinants are typically composed of chemically active surface typing (e.g., amino acids or sugar side chains) of molecules, and typically have specific three-dimensional structural features and specific charge characteristics. For example, antibodies can be produced for the N-terminal, central or C-terminal peptides of polypeptides. In addition, antibodies can be produced for the linear or interrupted epitopes of polypeptides. When the dissociation constant is ≤1 μM, for example, ≤100 nM in some embodiments and ≤10 nM in some embodiments, it is said that the antibody specifically binds to the antigen, and the antibody does not display the combination of closely related or different other proteins.

[0184] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and the antigen to which the immunoglobulin is specific. The strength or affinity of the immunological binding interaction can be measured by the dissociation constant (K) of the interaction. d ) indicates that K d The lower the affinity, the greater the affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complexing partner, the affinity of the interaction, and geometric parameters that affect the rates in both directions. Thus, the "association rate constant" (K) can be determined by calculating the concentration and the actual association and dissociation rates. on ) and the “dissociation rate constant” (K off (See Nature 361: 186-87 (1993)). K off / K on The ratio cancels all parameters not related to affinity and is equal to the dissociation constant K d (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) The binding constant (K) is determined as measured by an assay such as a radioligand binding assay or similar assays known to those skilled in the art. d ) is ≤1 μM, such as in some embodiments ≤100 nM, in some embodiments ≤10 nM, and in some embodiments ≤100 pM to about 1 pM, an antibody of the invention is said to specifically bind to EGFR.

[0185] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, by virtue of its origin, the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide wherein the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide wherein it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules encoding the heavy chain immunoglobulin molecules set forth herein and nucleic acid molecules encoding the light chain immunoglobulin molecules set forth herein.

[0186] As used herein, the term "isolated protein" means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which, by virtue of its source of origin or derivation, is (1) unrelated to proteins found in nature, (2) free of other proteins from the same source, e.g., free of murine proteins, (3) expressed by cells from a different species, or (4) not occurring in nature.

[0187] The term "polypeptide" is used herein as a general term to refer to a fragment or analog of a native protein, polypeptide sequence. Thus, native protein fragments and analogs are species of the polypeptide genus. Polypeptides according to the present disclosure include heavy chain immunoglobulin molecules as described herein and light chain immunoglobulin molecules as described herein, as well as antibody molecules comprising a combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule (e.g., a kappa light chain immunoglobulin molecule) and vice versa, as well as fragments and analogs thereof.

[0188] As used herein, the term "natural" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory or otherwise is natural.

[0189] As used herein, the term "operably linked" refers to the position of the components so described as being in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is joined in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0190] As used herein, the term "control sequence" refers to a polynucleotide sequence necessary for achieving expression and processing of the coding sequence to which it is joined. The nature of such control sequences varies depending on the host organism in prokaryotes, and such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences in eukaryotes. Typically, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide" as mentioned herein means nucleotides of at least 10 bases in length, ribonucleotides or deoxynucleotides, or a modified form of nucleotides of either type. The term includes DNA in both single-stranded and double-stranded forms.

[0191] The term oligonucleotide mentioned herein comprises the natural and modified nucleotides linked together by natural and non-natural oligonucleotide chains.Oligonucleotide generally comprises a polynucleotide subset of 200 or less base lengths.In some embodiments, oligonucleotide is 10 to 60 bases long, for example, in some embodiments, is 12,13,14,15,16,17,18,19 or 20 to 40 bases long.Oligonucleotide is normally a single strand such as for probes, but oligonucleotide can be a double strand such as for building a gene mutant.Oligonucleotide of the present disclosure is a sense or antisense oligonucleotide.

[0192] The term "natural nucleotides" mentioned herein include deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" mentioned herein include nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkage" mentioned herein includes oligonucleotide linkages, such as phosphorothioate, phosphorodithioate, selenophosphate, diselenphosphate, aniline phosphoric acid thiol ester (phosphoroanilothioate), aniline phosphate (phoshoraniladate), phosphoramidate (phosphoronmidate) etc. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucl. Acids Res. 16:3209 (1988), Zon et al., Anticancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford, England (1991); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). If desired, the oligonucleotide can include a label for detection.

[0193] As used herein, the 20 conventional amino acids and their abbreviations follow common usage. See Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the 20 conventional amino acids, non-natural amino acids (e.g., α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids) may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction, according to standard usage and convention.

[0194] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5′ end of the left-hand direction of a double-stranded polynucleotide sequence, referred to as the 5′ direction. The direction of 5′ to 3′ addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region on the DNA chain that has the same sequence as the RNA and is from the 5′ to 5′ end of the RNA transcript is referred to as the "upstream sequence," and the sequence region on the DNA chain that has the same sequence as the RNA and is from the 3′ to 3′ end of the RNA transcript is referred to as the "downstream sequence."

[0195] When applied to polypeptides, the term "substantially identical" means that two peptide sequences share at least 80 percent sequence identity, such as in some embodiments at least 90 percent sequence identity, in some embodiments at least 95 percent sequence identity, and in some embodiments at least 99 percent sequence identity, when optimally aligned, for example, by the programs GAP or BESTFIT using default gap weights.

[0196] In some embodiments, residue positions that differ differ by conservative amino acid substitutions.

[0197] As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as encompassed by the present invention, provided that the change in amino acid sequence maintains at least 75%, such as in some embodiments at least 80%, 90%, 95%, and in some embodiments 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those made within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamic acid, cysteine, serine, succinate, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamic acid, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are aliphatic-hydroxyl families; (ii) asparagine and glutamate, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, it is reasonable to expect that the isolated substitution of leucine with isoleucine or valine, the substitution of aspartate with glutamate, the substitution of threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve amino acids within framework sites. Whether an amino acid change results in a functional peptide can be readily determined by analyzing the specific activity of the polypeptide derivative. The analysis is described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. In some embodiments, the amino- and carboxyl-termini of the fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to published or proprietary sequence databases. Computerized comparison methods are used to identify sequence motifs or predicted protein conformational domains that occur in other proteins of known structure and / or function. Methods are known to identify protein sequences that fold into known three-dimensional structures. Bowie et al., Science 253:164 (1991). Thus, the above embodiments demonstrate that one skilled in the art can identify sequence motifs and structural conformations that can be used to define structural and functional domains according to the present disclosure.

[0198] In some embodiments, the amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs may include various muteins of sequences other than the native peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the native sequence (e.g., in portions of the polypeptide outside of the domains forming intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural characteristics of the parent sequence (e.g., the replacing amino acid should not tend to sever helices present in the parent sequence or disrupt other types of secondary structure that characterize the parent sequence). Examples of generally accepted polypeptide secondary and tertiary structure are set forth in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991).

[0199] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or one or more internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding positions in the inferred native sequence from, for example, a full-length cDNA sequence. A fragment is typically at least 5, 6, 8, or 10 amino acids long, for example, in some embodiments at least 14 amino acids long, in some embodiments at least 20 amino acids long, typically at least 50 amino acids long, and in some embodiments at least 70 amino acids long. As used herein, the term "analog" refers to a polypeptide comprising a segment of at least 25 amino acids, such amino acids having significant homology to a portion of the inferred amino acid sequence and specifically binding to EGFR under suitable binding conditions. Typically, a polypeptide analog comprises conservative amino acid substitutions (or additions or deletions) relative to the native sequence. Analogs are typically at least 20 amino acids long, for example, in some embodiments at least 50 amino acids long or longer, and typically can be as long as the full-length native polypeptide.

[0200] The term "pharmaceutical agent" is used herein to refer to a chemical compound, mixture of chemical compounds, biomacromolecule, or extract made from biological material.

[0201] As used herein, the term "label" or "labeled" refers to a polypeptide that incorporates a detectable marker, for example, by incorporating a radiolabeled amino acid or attaching to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). In some cases, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I), fluorescent labels (e.g., fluorophores, rhodamine, lanthanum phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labels are attached by spacers of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical agent or drug" refers to a chemical compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[0202] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and a substantially purified fraction is a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0203] Typically, a substantially pure composition will comprise greater than about 80%, for example, in some embodiments greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0204] The term patient includes human and veterinary subjects.

[0205] Other chemical terms are used herein according to customary usage in the industry as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. ed., McGraw-Hill, San Francisco (1985)).

[0206] II. Multispecific Polypeptide Constructs

[0207] Provided herein are multispecific polypeptide constructs comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein the first and second components are coupled by a linker, wherein the Fc region is located N-terminal to the CD3 binding region; and one or both of the first and second components comprise an antigen binding domain that binds a tumor-associated antigen (TAA).

[0208] In some embodiments, the multispecific polypeptide construct comprises, from N-terminus to C-terminus, in order: an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and an antigen binding domain that binds to a tumor-associated antigen (TAA). In some embodiments, the multispecific polypeptide construct comprises, from N-terminus to C-terminus, in order: an antigen binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; and a CD3 binding region that binds to CD3 (CD3ε). In some embodiments, the multispecific polypeptide construct contains at least a first antigen binding domain that binds to TAA and a second antigen binding domain that binds to TAA. In some embodiments, the multispecific polypeptide construct comprises, from N-terminus to C-terminus, in order: a first antigen binding domain that binds to a tumor-associated antigen (TAA); an immunoglobulin Fc region; a linker; a CD3 binding region that binds to CD3 (CD3ε); and a second antigen binding domain that binds to a tumor-associated antigen (TAA).

[0209] Each component of the multispecific polypeptide constructs of the present disclosure is described in more detail below.

[0210] 1. Anti-CD3 binding domain:

[0211] The multispecific polypeptide constructs of the present disclosure include one or more copies of anti-CD3 binding domains. The anti-CD3 binding domains of the present disclosure activate T cells via engagement of CD3ε on T cells. The anti-CD3 binding domains of the present disclosure excite, stimulate, activate and / or otherwise increase CD3-mediated T cell activation. The biological activities of CD3 include, for example, T cell activation and other signaling via interactions between CD3 and the antigen binding subunits of the T cell receptor (TCR). For example, the anti-CD3 binding domains of the present disclosure completely or partially activate T cells by regulating, for example, exciting, stimulating, activating or otherwise increasing CD3-mediated T cell activation via engagement of CD3ε on T cells.

[0212] In preferred embodiments, the anti-CD3 binding domains of the present disclosure specifically bind to the epsilon chain of CD3, also referred to as CD3ε. The anti-CD3ε binding domains of the present disclosure activate T cells by engaging CD3ε on T cells. The anti-CD3ε binding domains of the present disclosure include monoclonal antibodies, such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, and humanized monoclonal antibodies and chimeric antibodies, as well as antigen-binding fragments thereof. In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof.

[0213] In some embodiments, the anti-CD3ε binding domain comprises one or more copies of an antibody or antigen-binding fragment thereof selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the anti-CD3 binding domain comprises an Fv antibody fragment that binds to CD3ε (referred to herein as an anti-CD3ε Fv fragment). In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3 binding domain monovalently binds to CD3.

[0214] In some embodiments, the anti-CD3 epsilon binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 14 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 44 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3 epsilon binding domain comprises a combination of a heavy chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 32-81 and a light chain variable region amino acid sequence. In some embodiments, the anti-CD3 epsilon binding domain comprises a combination of a heavy chain variable region amino acid sequence selected from the group of SEQ ID NOs: 32-62 and a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group of SEQ ID NOs: 63-81.

[0215] In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD3ε binding domain comprises a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14 and a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15.

[0216] In some embodiments, the anti-CD3 epsilon binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-CD3 epsilon binding domain comprises a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD3ε binding domain comprises a variable heavy chain (Hv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 44 and a variable light chain (Lv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 72.

[0217] In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε Fv antibody fragment comprises a combination of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0218] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CD2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

[0219] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence comprising at least the amino acid sequence GTNCRAP (SEQ ID NO: 20); and a VLCDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

[0220] In some embodiments, the anti-CD3ε binding domain comprises a VH CDR1 sequence comprising at least the amino acid sequence of TYAMN (SEQ ID NO: 16); a VHCD2 sequence comprising at least the amino acid sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence comprising at least the amino acid sequence of HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence comprising at least the amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence comprising at least the amino acid sequence of GTNCRAP (SEQ ID NO: 20); and a VLCDR3 sequence comprising at least the amino acid sequence of ALWYSNLWV (SEQ ID NO: 21).

[0221] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CD2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and a VH CD2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: NO: 18) VHCDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

[0222] In some embodiments, the anti-CD3ε binding domain comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of GTNCRAP (SEQ ID NO: 20); and a VL CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of ALWYSNLWV (SEQ ID NO: NO: 21) VL CDR3 sequences having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical.

[0223] In some embodiments, the anti-CD3ε binding domain comprises a VHCDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TYAMN (SEQ ID NO: 16); a VH CD2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of HGNFGNSYVSWFAY (SEQ ID NO: 18); a VH CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 19); NO:19); a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of GTNCRAP (SEQ ID NO:20); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of ALWYSNLWV (SEQ ID NO:21).

[0224] In some embodiments, the anti-CD3ε binding domain thereof is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain thereof is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81 and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain thereof is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-81 and a light chain variable amino acid sequence. In some embodiments, the anti-CD3ε binding domain thereof is an Fv fragment comprising a combination of a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81. In some embodiments, the anti-CD3ε binding domain thereof is an Fv fragment comprising a heavy chain variable amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-62, and an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-81.

[0225] 2. Immunoglobulin Fc polypeptide:

[0226] The first component of the multispecific polypeptide constructs of the present disclosure comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 subclass. In some embodiments, the Fc region is a human Fc. In some embodiments, the immunoglobulin Fc region is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region comprises an Fc chain that is an immunologically active fragment of any one of SEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region comprises an Fc polypeptide chain or immunologically active fragment thereof that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0227] In some embodiments, a multispecific polypeptide construct is a dimer formed from polypeptides that each contain an Fc region. In some embodiments, identical or substantially identical polypeptides dimerize to produce a homodimer. In some embodiments, a dimer is a homodimer in which the two polypeptides of the multispecific polypeptide construct are identical. In other cases, the Fc region is formed from an Fc domain that has been mutated or modified to promote heterodimerization, in which different polypeptides can dimerize to produce a heterodimer. Thus, in some embodiments, a dimer is a heterodimer in which the two polypeptide chains of the multispecific polypeptide construct are different. Exemplary modifications known to promote heterodimerization include any of the following.

[0228] In general, in addition to antigen binding ability, which is the primary function of immunoglobulins, the Fc region is also responsible for effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Furthermore, the FcRn sequence present in the Fc region plays a role in regulating IgG levels by extending half-life in vivo through coupling to the FcRn receptor in vivo. In some embodiments, such functions can be altered, e.g., reduced or enhanced, when using the Fc region of the provided multispecific polypeptide constructs.

[0229] In some embodiments, the Fc region of the provided multispecific polypeptide constructs exhibits one or more effector functions. In some cases, the Fc region can provide Fc-mediated effector functions, such as ADCC (e.g., release of granzyme B by NK cells), ADCP, and / or CDC. Thus, in some embodiments in which the multispecific polypeptide constructs contain a cleavable linker, cleavage of the linker can produce two components, each with biological activity: a CD3 binding region capable of binding and engaging CD3 on T cells, and an Fc region linked to a TAA-antigen binding domain that can exhibit target-specific effector functions.

[0230] In some embodiments, the Fc region comprises an Fc polypeptide that has been mutated or modified to alter one or more effector functions. Various embodiments of mutating an Fc polypeptide to alter (e.g., reduce) effector function are known, including any of the embodiments described below. In some embodiments, unless otherwise described with reference to a specific SEQ ID NO, reference to amino acid substitutions in the Fc region is by EU numbering as in Kabat (also referred to as Kabat numbering). EU numbering is known and is based on the most recently updated IMGTScientific Chart ( the international ImMunoGeneTics information http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (Created May 17, 2001, Last updated January 10, 2013) and the EU index as reported in: Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0231] In some embodiments, provided multispecific polypeptide constructs containing Fc regions that exhibit reduced effector functions may be desirable candidates for applications where limited CD3 binding is desired but certain effector functions (e.g., CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the multispecific polypeptide construct and / or its cleaved components have no FcγR binding (and therefore may have no ADCC activity), but retain FcRn binding ability. Primary cells used to mediate ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (e.g., see ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.) and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the multispecific polypeptide construct and / or its cleaved components are unable to bind C1q and therefore lack CDC activity. For example, see WO 2006 / 029879 and WO 2005 / 100402 for C1q and C3c binding ELISAs. To assess complement activation, a CDC assay can be performed (e.g., see Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (e.g., see Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0232] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof is of the IgG isotype. For example, the immunoglobulin Fc region of the fusion protein is of the human IgG 1 isotype, which has the following amino acid sequence:

[0233]

[0234] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgG1 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0235] In some embodiments, the human IgG1 Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al., 1992 J Immunol, 148: 3461-3468; see Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11, the entire contents of which are incorporated herein by reference.

[0236] In some embodiments, the Fc region (e.g., human IgG1 Fc region) is modified to enhance ADCC activity or CDC activity. Embodiments of mutations that enhance ADCC include modifications of Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications of Lys326 and Glu333. In some embodiments, the Fc region is modified at one or two of these positions using the Kabat numbering system, such as Lys326Ala and / or Glu333Ala (K326A and E333A).

[0237] In some embodiments, the human IgG1 Fc-region fusion proteins of the present invention have no or reduced fucose attached to N-linked glycan chains at N297. There are various ways to prevent fucosylation, including, but not limited to, production in FUT8-deficient cell lines; addition of inhibitors, such as castanospermine, to mammalian cell culture media; and metabolic engineering of production cell lines. In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the fusion protein, such as Asn297Ala (N297A) or Asn297Asp (N297D).

[0238] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu 235 (L235), Asp 265 (D265), Asp 270 (D270), Ser 298 (S298), Asn 297 (N297), Asn 325 (N325), or Ala 327 (A327). For example, Leu 234 Ala (L234A), Leu 235 Ala (L235A), Asp 265 Asn (D265N), Asp 270 Asn (D270N), Ser 298 Asn (S298N), Asn 297 Ala (N297A), Asn 325 Glu (N325E), or Ala 327 Ser (A327S). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (boxed in SEQ ID NO: 1 above, Kabat numbering) to alter Fc receptor interactions, such as Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (boxed in SEQ ID NO: 1 above, Kabat numbering) to alter Fc receptor interactions, such as Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234 and 235, such as Leu234A1a and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In preferred embodiments, modifications within the Fc region reduce binding to the Fc-receptor-gamma receptor while having minimal effect on binding to the neonatal Fc receptor (FcRn).

[0239] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Embodiments of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256G1u (respectively M252Y, S254T, T256E) (Kabat numbering, Dall'Acqua et al., 2006, J.Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al., 2010 Nature Biotech, Vol. 28(2), 157-159) (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the mutated or modified Fc polypeptide comprises the following mutations: using the Kabat numbering system, Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L or M252Y, M428V).

[0240] In some embodiments, the Fc region of the fusion protein lacks amino acids at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In such embodiments, the Fc deletion of these three amino acids reduces complement protein C1q binding.

[0241] PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPREEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQVSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 2)

[0242] In some embodiments, the Fc region of the fusion protein is altered at Gly236 (boxed in SEQ ID NO: 1 above) to reduce Fc receptor binding. For example, G1y236 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A, such as Gly236A1a (G236A).

[0243] In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0244] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2.

[0245] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG2 isotype and has the following amino acid sequence:

[0246]

[0247] In some embodiments, the fusion or immunologically active fragment thereof comprises a human IgG2 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:3.

[0248] In some embodiments, the human IgG2 Fc region is modified at amino acid Asn297 (boxed) to prevent glycosylation of the antibody, such as Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG2 Fc region lacks Lys447 (EU index of Kabat et al. 1991, Sequences of Proteins of Immunological Interest).

[0249] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG3 isotype and has the following amino acid sequence:

[0250]

[0251]

[0252] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG3 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4.

[0253] In some embodiments, the human IgG3 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend half-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al. 1991, Sequences of Proteins of Immunological Interest).

[0254] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the following amino acid sequence:

[0255]

[0256] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5.

[0257] In some embodiments, the immunoglobulin Fc region or immunologically active fragment of the fusion protein is of the human IgG4 isotype and has the following amino acid sequence:

[0258]

[0259]

[0260] In some embodiments, the antibody or immunologically active fragment thereof comprises a human IgG4 polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6.

[0261] In other embodiments, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, such as Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (boxed, Kabat numbering) to prevent glycosylation of the antibody, such as Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al. 1991, Sequences of Proteins of Immunological Interest).

[0262] In some embodiments, the human IgG Fc region is modified to stabilize homodimerization by introducing two disulfide bonds on the CH3:CH3 surface by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) (S354C / Y349C).

[0263] In some embodiments, the human IgG Fc region is modified to induce heterodimerization. Various methods for promoting heterodimerization of complementary Fc polypeptides are known, for example, see Ridgway et al., Protein Eng. 9: 617-621 (1996); Merchant et al., Nat. Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3: 546-57; Von Kreudenstein et al. MAbs, (2013) 5: 646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285: 19637-46; Leaver-Fay et al. (2016) Structure, 24: 641-51; Ha et al. (2016) Frontiers in Immunology, 7: 1; Davis et al. (2010) Protein Eng Des Se 1, 23: 195-202; Published International PCT Invention No. WO No. 1998 / 050431, No. WO 2009 / 089004, No. WO2011143545, No. WO2014 / 067011, No. WO 2012 / 058768, No. WO2018027025; published U.S. patent invention No. US20140363426, No. US20150307628, No. US20180016354, No. US20150239991; and U.S. Patent No. US5731168, No. US7183076, No. US9701759, No. US9605084 and No. US9650446. Methods for promoting heterodimerization of Fc chains include mutagenesis of the Fc region, for example, by including a set of "knob-in-hole" mutations or mutations to achieve electrostatic manipulation of the Fc to favor attractive interactions between different polypeptide chains. For example, in some embodiments, the heterodimeric Fc polypeptides include mutations to change the charge polarity across the surface of the Fc dimer, such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions inhibit the formation of undesirable Fc homodimers (Guneskaran et al. (2010) JBC, 285: 19637-19646). When co-expressed in cells, interchain association is possible, but due to charge repulsion, such chains do not substantially self-associate. Other strategies for generating heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to produce complementary CH3 heterodimers, which are called SEED Fc.

[0264] In some embodiments, to promote heterodimerization, both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary paired amino acid modifications of the polypeptides of the Fc fusion are described in Table 1.

[0265]

[0266] In some embodiments, the modification comprises introducing a protrusion (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide, such that the protrusion can be positioned within the cavity to facilitate complexing of the first and second Fc-containing polypeptides. The amino acids targeted for replacement and / or modification to create a protrusion or cavity in a polypeptide are typically surface amino acids that interact or contact one or more amino acids on the surface of the second polypeptide.

[0267] In some embodiments, the first Fc polypeptide modified to contain a protrusion (hole) amino acid comprises replacing a natural or original amino acid with an amino acid having at least one side chain that protrudes from the surface of the first Fc polypeptide and can therefore be positioned in a complementary cavity (hole) in the adjacent surface of the second polypeptide. Most often, the replacement amino acid is one with a side chain volume larger than the original amino residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those that are ideal replacement amino acids to produce a protrusion. In some embodiments, the replacement residues used to form the protrusion are natural amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W). In some embodiments, the original residue identified for replacement is an amino acid residue with a small side chain, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0268] In some embodiments, the second Fc polypeptide modified to contain a cavity (hole) comprises replacing a natural or original amino acid with an amino acid having at least one side chain that is recessed from the surface of the second polypeptide and is thus able to accommodate a corresponding protrusion from the surface of the first polypeptide. Most often, the replacement amino acid is one that has a smaller side chain volume than the original amino residue. Those skilled in the art know how to determine and / or evaluate the properties of amino acid residues to identify those that are ideal replacement amino acids for forming a cavity. Typically, replacement residues for forming a cavity are natural amino acid residues and include, for example, alanine (A), serine (S), threonine (T), and valine (V). In some embodiments, the original amino acid identified for replacement is an amino acid with a large side chain, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0269] The CH3 surface of human IgG1, for example, comprises 16 residues on each domain, which are located on four antiparallel β-strands and are buried at a distance from each surface. (See, e.g., Deisenhofer et al. (1981) Biochemistry, 20: 2361-2370; Miller et al. (1990) J Mol. Biol., 216, 965-973; Ridgway et al. (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168.) Modifications of the CH3 domain to create a protrusion or cavity are described, e.g., in U.S. Pat. No. 5,731,168; International Patent Inventions WO98 / 50431 and WO2005 / 063816; and Ridgway et al. (1996) Prot. Engin., 9: 617-621. In some embodiments, modifications of the CH3 domain to create a protrusion or cavity are typically targeted to residues located on the two central antiparallel β strands. The aim is to minimize the risk that the resulting protrusion may be accommodated by protruding into the surrounding solvent rather than being accommodated by a complementary cavity in the partner CH3 domain.

[0270] For example, in some embodiments, a heterodimeric Fc comprises a polypeptide having an amino acid modification at Thr366 within the CH3 domain, which, when replaced with a larger amino acid (e.g., Try (T366W)), preferentially pairs with a second CH3 domain having amino acid modifications at positions Thr366, Leu368, and Tyr407 that are smaller (e.g., Ser, Ala, and Val (T366S / L368A / Y407V), respectively). Heterodimerization via CH3 modifications can be further stabilized by introducing disulfide bonds, for example, by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on opposing CH3 domains (see Carter, 2001 Journal of Immunological Methods, 248:7-15).

[0271] The resulting multispecific polypeptide construct can be purified by any suitable method, for example, by affinity chromatography on a Protein A or Protein G column. If two nucleic acid molecules encoding different polypeptides are transformed into cells, homodimers and heterodimers will form. Expression conditions can be adjusted to favor heterodimer formation over homodimer formation.

[0272] Techniques for recovering heterodimers from homodimers based on their differential affinity for affinity reagents are known. In some aspects, such techniques include designing heterodimers so that one of the Fc polypeptide chains does not bind to the affinity reagent Protein A. In some cases, one of the polypeptide chains may contain one or more amino acid substitutions to eliminate or reduce the affinity of one of the polypeptides of the Fc heterodimer for the Protein A reagent, see, for example, WO2017134440, WO2010151792, Jendeberg et al. (Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some of such embodiments, the Fc region may be modified at the protein-A binding site on one member of the heterodimer to prevent protein-A binding and thereby be more effective. Effectively purify heterodimeric fusion proteins. An exemplary modification within this binding site is Ile253, such as Ile253Arg (I253R). In some embodiments, the modification may be H435R or H435R / Y436F. In some embodiments, the Fc polypeptide of the Fc heterodimer may contain modifications to enable it to bind protein A rather than protein G (pA+ / pG-). Exemplary pA+ / pG- amino acid modifications include Fc containing serine at position 428, serine at position 434, and optionally histidine at position 436, with reference to human IgG1 or comprising such residues at the corresponding positions of human IgG 2, 3, or 4. In some aspects, such amino acid modifications in an IgG Fc polypeptide at positions 428, 434, and optionally 436 reduce or prevent the binding of protein G, thereby enhancing the purification of the protein.

[0273] In some embodiments, any such modification that imparts differential affinity to an affinity reagent can be combined with any one or more of the other amino acid modifications described above. For example, the I253R modification can be combined with the T366S / L368A / Y407V modification or the T366W modification. An Fc modified with T366S / L368A / Y407V can form homodimers due to the lack of steric closure of the dimerization surface, as in the case of an Fc modified with T366W. Therefore, in some embodiments, the I253R modification is combined with an Fc modified with T366S / L368A / Y407V to prevent purification of any homodimeric Fc that may form. Similar modifications can be utilized by combining T366S / L368A / Y407V and H453R.

[0274] In some embodiments, the Fc region of the heterodimeric molecule may additionally contain one or more other Fc mutations, such as any of those described above. In some embodiments, the heterodimeric molecule contains an Fc region with a mutation that reduces effector function.

[0275] In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 82, 86, 94, or 96, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 87, 90, 92, 98, or 100. In some embodiments, one Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 84, 88, 95, or 97, and the other Fc polypeptide of the heterodimeric Fc comprises the amino acid sequence set forth in any one of SEQ ID NOs: 85, 89, 93, 99, or 101.

[0276] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In such embodiments, the fusion protein of the present invention is monomeric. For example, modification of residue Thr366 to a charged residue (e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively)) prevents CH3-CH3 dimerization.

[0277] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu 235 (L235), Asp 265 (D265), Asp 270 (D270), Ser 298 (S298), Asn 297 (N297), Asn 325 (N325), or Ala 327 (A327). For example, Leu 234 Ala (L234A), Leu 235 Ala (L235A), Asp 265 Asn (D265N), Asp 270 Asn (D270N), Ser 298 Asn (S298N), Asn 297 Ala (N297A), Asn 325 Glu (N325E), or Ala 327 Ser (A327S). In a preferred embodiment, modifications within the Fc region reduce binding to the Fc-receptor-gamma receptor while having minimal effect on binding to the neonatal Fc receptor (FcRn).

[0278] In some embodiments, the fusion protein contains a polypeptide derived from an immunoglobulin hinge region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein can contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 7), in which Cys220, which forms a disulfide with the C-terminal cysteine ​​of the light chain, is mutated to a serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 8).

[0279] In some embodiments, the fusion protein has a modified hinge from IgG4 that has been modified to prevent or reduce chain changes, such as Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 9). In some embodiments, the fusion protein contains a linker polypeptide. In other embodiments, the fusion protein contains both a linker and a hinge polypeptide.

[0280] 3. Connector:

[0281] The multispecific polypeptide constructs provided contain a linker that connects or couples a first component containing an immunoglobulin Fc region and a second component containing a CD3 binding region. In some embodiments, the linker is located at the end of the C-terminal region of the Fc region, so that the Fc region is at the N-terminal end of the CD3 binding region. Since the multispecific polypeptide constructs provided are multimers (e.g., dimers), the constructs provided include a linker that connects the first Fc polypeptide to the first domain (e.g., VH) of the CD3 binding region of the first polypeptide and the second Fc polypeptide to the second domain (e.g., VL) of the CD3 binding region of the second polypeptide. Typically, the linker present in the first and second polypeptides of the multispecific polypeptide construct is the same. Therefore, in some embodiments, each domain of the CD3 binding domain is connected to the opposite polypeptide of the Fc (e.g., a heterodimeric Fc) via a linker (e.g., the same linker).

[0282] Various polypeptide linkers are known for use in fusion proteins (e.g., see Chen et al. (2013) Adv. Drug. Deliv. 65: 1357-1369; and International PCT Publication Nos. WO 2014 / 099997, WO 2000 / 24884; U.S. Pat. No. 5,258,498; U.S. Pat. No. 5,525,491; U.S. Pat. No. 5,525,491, U.S. Pat. No. 6,132,992).

[0283] In some embodiments, the linker is selected such that when the CD3 binding region is linked to the Fc region of the multispecific polypeptide conjugate, the CD3 binding region is restricted and is unable or substantially unable to bind or engage CD3 on the surface of a cell (e.g., a T cell) when the multispecific polypeptide construct is in contact with the cell. Various assays can be used to evaluate the binding or engagement of CD3 by the multispecific polypeptide construct, including assays for evaluating T cell binding, NFAT activation using reporter gene systems, cytolytic T cell activity, cytokine production, and / or expression of T cell activation markers. Exemplary assays are shown in the provided Examples. Generally, the linker also ensures that the polypeptide construct folds correctly, does not exhibit a charge that would be inconsistent with the activity or function of the linked polypeptide, or does not form bonds or other interactions with amino acid residues in one or more domains that could hinder or alter the activity of the linked polypeptide. In some embodiments, the linker is a polypeptide linker. The polypeptide linker can be a flexible linker or a rigid linker, or a combination of both. In some aspects, the linker is a short, medium, or long linker. In some embodiments, the linker is up to 40 amino acids long. In some embodiments, the linker is up to 25 amino acids long. In some embodiments, the linker is at least or is at least about 2 amino acids long. In some aspects, a suitable length is, for example, at least one and typically less than about 40 amino acid residues (e.g., 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acids) long. In some embodiments, the linker is about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.

[0284] In certain aspects, the longer the linker length, the stronger the CD3 binding when the multispecific polypeptide conjugate is bound to its antigen (e.g., TAA). Thus, in some aspects, the linker is greater than 12 amino acids in length, e.g., greater than 13, 14, 15, 16, 17, or 18 amino acids in length. In some embodiments, the linker is 12 to 40 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30 amino acids, or 30 to 40 amino acids in length.

[0285] The linker can be natural, synthetic or a combination of the two. Particularly suitable linker polypeptides mainly include amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala) and threonine (Thr). For example, the linker can contain at least 75% (calculated based on the total number of residues present in the peptide linker), such as at least 80%, at least 85% or at least 90% amino acid residues selected from Gly, Ser, Ala and Thr. The linker can also consist only of Gly, Ser, Ala and / or Thr residues. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues or 8-12 glycine residues. In some aspects, suitable peptide linkers generally contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, the peptide linker only comprises glycine residues. In some embodiments, the peptide linker only comprises glycine and serine residues.

[0286] In some embodiments, such linkers are primarily composed of the amino acids glycine and serine, and are denoted herein as GS-linkers. In some embodiments, the linker comprises (GGS)n, wherein n is 1 to 10, such as 1 to 5, such as 1 to 3, such as GGS(GGS)n (SEQ ID NO: 171), wherein n is 0 to 10. In a specific embodiment, the linker comprises the sequence (GGGGS)n (SEQ ID NO: 173), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3. In other embodiments, the linker comprises (GGGGGS)n (SEQ ID NO: 172), wherein n is 1 to 4, such as 1 to 3. The linker may comprise a combination of any of the above, for example, 2, 3, 4, or 5 repeats of GS, GGS, GGGGS, and / or GGGGGS linkers may be combined. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0287] In some embodiments, the linker is (in single-letter amino acid code): GGS, GGGGS (SEQ ID NO: 149) or GGGGGS (SEQ ID NO: 135). In some embodiments, the GS-linker comprises the following amino acid sequence: GGSGGS, also known as (GGS)2 (SEQ ID NO: 10); GGSGGSGGS, also known as (GGS)3 (SEQ ID NO: 11); GGSGGSGGSGGS, also known as (GGS)4 (SEQ ID NO: 12); GGSGGSGGSGGSGGS, also known as (GGS)5 (SEQ ID NO: 13); GGGGGSGGGGSGGGGGS, also known as (G5S)3 (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147) and GGGGSGGGGSGGGGS (SEQ ID NO: 170). In some embodiments, the linker is GGGG (SEQ ID NO: 103). In some of any of the above embodiments, the serine can be replaced with an alanine (eg, (Gly4Ala) or (Gly3Ala)).

[0288] In some embodiments, the linker comprises a linker having the amino acid sequence Gly x Xaa-Gly y -Xaa-Gly z (SEQ ID NO: 174), wherein each Xaa is independently selected from alanine (A1a), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamic acid (Gln), lysine (Lys), arginine (Arg), histidine (His), aspartate (Asp), and glutamate (Glu), and wherein x, y, and z are each an integer in the range of 1-5. In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a specific variation, each of x, y, and z is equal to 3 (thereby generating a peptide linker having the amino acid sequence Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO: 175), where each Xaa is selected as above.

[0289] In some embodiments, the linker is a serine-rich linker based on repetitions of the (SSSSG)y (SEQ ID NO: 185) motif, where y is at least 1, but y can be 2, 3, 4, 5, 6, 7, 8, and 9.

[0290] In some cases, it may be desirable to provide some rigidity to the peptide linker. This can be accomplished by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, the peptide linker may have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In a specific embodiment, the peptide linker comprises only proline residues.

[0291] In some aspects, the peptide linker comprises at least one cysteine ​​residue, such as a cysteine ​​residue. For example, in some embodiments, the linker comprises at least one cysteine ​​residue and an amino acid residue selected from the group consisting of Gly, Ser, Ala and Thr. In some such embodiments, the linker comprises a glycine residue and a cysteine ​​residue, such as only a glycine residue and a cysteine ​​residue. Typically, each peptide linker will include only one cysteine ​​residue. An example of a specific linker comprising a cysteine ​​residue includes a linker having the amino acid sequence Gly m -Cys-Gly n wherein n and m are each an integer from 1 to 12, such as 3 to 9, 4 to 8, or 4 to 7. In a specific variation, the peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO: 177).

[0292] In some embodiments, the linker of the fusion protein is a structured or constrained linker. In a specific embodiment, the structured linker contains the sequence (AP)n or (EAAAK)n (SEQ ID NO: 178), wherein n is 2 to 20, preferably 4 to 10, including but not limited to AS-(AP)n-GT (SEQ ID NO: 179) or AS-(EAAAK)n-GT (SEQ ID NO: 180), wherein n is 2 to 20, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments, the linker comprises the sequence (GGGGA)n (SEQ ID NO: 181), (PGGGS)n (SEQ ID NO: 182), (AGGGS)n (SEQ ID NO: 183), or GGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 184, wherein n is 2 to 20. In some embodiments, the linker is SSSASASSA (SEQ ID NO: 186), GSPGSPG (SEQ ID NO: 187), or ATTTGSSPGPT (SEQ ID NO: 176). In some embodiments, such linkers may be more resistant to proteolytic degradation due to their structure, thereby providing advantages during in vivo injection.

[0293] In some embodiments, the joint is not a cleavable joint, also referred to as a non-cleavable joint. In some embodiments, the joint cannot be cleaved by a protease. In some embodiments, the joint that is not a cleavable joint or cannot be cleaved by a protease is generally stable for in vivo delivery or recombinant production. In some aspects, the joint that cannot be cleaved by a protease includes those that do not contain at least one peptide bond, and the peptide bond is preferably located within a cleavable peptide sequence or recognition site of a protease. In a specific embodiment, the non-cleavable joint is not a target substrate of a protease, so that compared to a joint containing a substrate recognition site of the same protease, it is not preferentially or specifically cleaved by a protease.

[0294] In some embodiments, the linker is a cleavable linker. In some aspects, a cleavable linker is a linker comprising a sequence that is a substrate for a protease, due to the presence of at least one bond that can be broken under physiological conditions. In some cases, a cleavable linker is susceptible to cleavage or is sensitive to cleavage under specific conditions present in vivo, such as after exposure to extracellular proteases (including those present in vivo in the cellular environment). In some cases, proteases may be present in specific physiological microenvironments, such as tumor microenvironments, thereby limiting the sites where cleavage may occur.

[0295] Compared to another non-target substrate, a protease typically exhibits specificity or preference for the cracking of a specific target substrate. The degree of this specificity can be determined based on the cleavage rate constant of a sequence (e.g., a joint), which is a measure of the preference of a protease for its substrate and the efficiency of the enzyme. Any method for determining the rate at which cracking increases over time in the presence of substrates at varying concentrations can be used to calculate the specificity constant. For example, a substrate is linked to a fluorescent probe portion that is released upon cleavage by a protease. By measuring the cleavage rate at different protease concentrations, the cleavage specificity constant (k) for a specific protease to a specific joint can be determined. cat / K m In some embodiments, the cleavable linker is a cleavable linker that can be cleaved at a rate of about at least 1×10 4 M -1 S -1 , or at least 5×10 4 M -1 S, at least 10×10 4 M -1 S, at least 10×10 5 M -1 Linkers that are specifically cleaved by proteases at a rate of 5s or greater.

[0296] Cleavable linker

[0297] In some embodiments, the multi-specific polypeptide constructs of the present disclosure include a cleavable linker that connects the first and second components. In some embodiments, the cleavable linker includes an amino acid sequence that can be used as a substrate for a protease, typically an extracellular protease. For example, the cleavable linker may include a cleavage sequence containing at least one peptide bond that is located within the cleavable peptide sequence of the protease. Suitable proteases include, for example, matrix metalloproteinases (MMPs), cysteine ​​proteases, serine proteases, and plasmin activators, which are formed or activated in an enhanced manner in diseases such as rheumatoid arthritis or cancer, leading to excessive tissue degradation, inflammation, and metastasis. In specific embodiments, the protease is a protease produced by cells in a tumor, activated immune effector cells (e.g., T cells or NK cells), or a tumor microenvironment. In some embodiments, the protease is granzyme B, an interstitial protease, or an MMP (e.g., MMP-2).

[0298] Cleavable linkers can be selected based on proteases produced by tumors in close proximity to cells expressing the target and / or produced by tumors in tissues co-localized with the desired target of the multispecific polypeptide construct. Increased levels of proteases with known substrates have been reported in many cancers (e.g., solid tumors). See, e.g., La Rocca et al., (2004) British J. of Cancer 90(7): 1414-1421.

[0299] In some embodiments, the cleavable linker connecting the first and second components of the multispecific polypeptide construct can be cleaved by a protease produced by an immune effector cell that is activated by one of these components. For example, a multispecific polypeptide construct encompassing an effector-activated or enhanced IgG Fc region can trigger ADCC when engaged with a target antigen. Crucial to ADCC is the release of granzyme B and perforin from effector cells (i.e., NK cells and cytotoxic T cells). Upon release, granzyme B enters the target cell in a perforin-dependent manner, where it mediates apoptosis. Importantly, granzyme B is active within the extracellular synapse between the effector cell and the target cell. In some embodiments, the cleavable linker connecting the first and second components of the multispecific polypeptide construct can be cleaved by granzyme B. During effector cell activation mediated by one of the components of the multispecific polypeptide construct, granzyme B is released. In some embodiments, granzyme B and other proteases can be produced by immune effector cells (including activated T cells or NK cells). In some embodiments, upon TAA binding by the multispecific polypeptide construct, activation of T cells through CD3 engagement can release such proteases, which can then cleave the specific cleavable linker, thereby enhancing or increasing the activity of the CD3 binding molecule in binding to CD3. In some embodiments, cleavage can amplify or increase the activity achieved by the multispecific construct when bound to the TAA in the uncleaved state.

[0300] Exemplary substrates include, but are not limited to, substrates that can be cleaved by one or more of the following enzymes or proteases: ADAMS, ADAMTS, such as ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartate proteases, such as BACE or renin; aspartate autolytic enzymes, such as autolytic enzyme D or autolytic enzyme E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14; cysteine ​​autolytic enzymes, such as autolytic enzyme B, autolytic enzyme C, autolytic enzyme K, autolytic enzyme L, autolytic enzyme S, autolytic enzyme V / L2, autolytic enzyme X / Z / P; cysteine ​​proteases, such as kumarase; asparagine endopeptidase; Otubain-2; KLK, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK LK14; metalloproteinases, such as meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27; serine proteases, such as activated protein C, lysin A, lysin G, chymosin, coagulation factor proteases (e.g., Such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoate esterase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, plasmin, uPA; type II transmembrane serine protease (TTSP), such as DESC1, DPP-4, FAP, Hepsin, intercalated proteinase-2, intercalated proteinase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof.

[0301] In some embodiments, the cleavable linker can be cleaved by multiple proteases, e.g., 2 or more proteases, 3 or more proteases, 4 or more proteases, etc.

[0302] In some embodiments, the cleavable linker is selected using a specific protease, such as a protease known to be produced by tumors in close proximity to cells expressing the target and / or produced by tumors that co-localize with the target of the multispecific polypeptide construct.

[0303] In some embodiments, the cleavable linker contains a substrate recognition site or cleavage site for a specific protease, which is a sequence recognized by the active site of the protease cleaved by the protease. Typically, for example, for serine proteases, the cleavage sequence is composed of the P1-P4 and P1'-P4' amino acids in the substrate, wherein cleavage occurs after the P1 position. Typically, the cleavage sequence of serine proteases is 6 residues long to match the expanded substrate specificity of many proteases, but can be longer or shorter depending on the protease. Typically, the cleavable linker includes a P1-P1' scissile bond sequence recognized by the protease. In some aspects, the cleavable linker is modified to introduce a peptide bond that can be cleaved by a specific protease, for example, by introducing a substrate recognition site sequence or cleavage sequence of a protease.

[0304] In some embodiments, the cleavable linker comprises a combination of two or more substrate sequences. In some embodiments, each substrate sequence is cleaved by the same protease. In some embodiments, at least two of the substrate sequences are cleaved by different proteases. In some embodiments, the cleavable linker comprises amino acids that are substrates for granzyme B. In some embodiments, the granzyme B cleavable linker comprises an amino acid sequence having the general formula P4 P3 P2 P1 ↓P1' (SEQ ID NO: 150), wherein P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A. In some embodiments, the granzyme B cleavable linker comprises an amino acid sequence having the general formula P4 P3 P2 P1↓P1' (SEQ ID NO: 151), wherein P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G.

[0305] In some embodiments, the substrate for granzyme B comprises the amino acid sequence LEAD (SEQ ID NO: 22), LEPG (SEQ ID NO: 142), or LEAE (SEQ ID NO: 143). In some embodiments, the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO: 136), LEPDG (SEQ ID NO: 152), LEADT (SEQ ID NO: 137), IEPDG (SEQ ID NO: 138), IEPDV (SEQ ID NO: 139), IEPDS (SEQ ID NO: 140), IEPDT (SEQ ID NO: 141), IEPDP (SEQ ID NO: 144), LEPDG (SEQ ID NO: 152), or LEADG (SEQ ID NO: 153).

[0306] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for an interstitial protease. In some embodiments, the cleavable linker comprises the sequence P4QAR↓(A / V) (SEQ ID NO: 154), where P4 is any amino acid. In some embodiments, the cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155). In some embodiments, the substrate for an interstitial protease comprises the amino acid sequence RQAR (SEQ ID NO: 23). In some embodiments, the cleavable linker comprises the amino acid sequence RQARV (SEQ ID NO: 156).

[0307] In some embodiments, the cleavable linker comprises an amino acid that is a substrate for one or more matrix metalloproteinases (MMPs). In some embodiments, the MMP is MMP-2. In some embodiments, the cleavable linker comprises the formula P3 P2 P1 ↓ P1' (SEQ ID NO: 157), wherein P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1' is L, I, or M. In some embodiments, the cleavable linker comprises the formula P3 P2 P 1 ↓ P1' (SEQ ID NO: 158), wherein P3 is P; P2 is Q or D; P1 is A or N; and P1' is L or I. In some embodiments, the substrate for the MMP comprises the amino acid sequence PAGL (SEQ ID NO: 24).

[0308] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for a stromal protease. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 22) and the amino acid sequence RQAR (SEQ ID NO: 23).

[0309] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 22) and the amino acid sequence PAGL (SEQ ID NO: 24).

[0310] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for a stroma protease and an amino acid sequence that is a substrate for a MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence RQAR (SEQ ID NO: 23) and the amino acid sequence PAGL (SEQ ID NO: 24).

[0311] In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B, an amino acid sequence that is a substrate for a streptococcus protease, and an amino acid sequence that is a substrate for an MMP. In some embodiments, the cleavable linker comprises a combination of an amino acid sequence that is a substrate for granzyme B and an amino acid sequence that is a substrate for an MMP. In some embodiments, the cleavable linker comprises a combination of the amino acid sequence LEAD (SEQ ID NO: 22), the amino acid sequence RQAR (SEQ ID NO: 23), and the amino acid sequence PAGL (SEQ ID NO: 24).

[0312] Cleavable linkers may include any known linker. Examples of cleavable linkers are described in Be'liveau et al. (2009) FEBS Journal, 276; U.S. Patent Publication Nos. US20160194399; US20150079088; US20170204139; US20160289324; US20160122425; US20150087810; US20170081397; U.S. Patent No. US9644016.

[0313] In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of: TGLEADGSPAGLGRQARVG (SEQ ID NO: 25); TGLEADGSRQARVGPAGLG (SEQ ID NO: 26); TGSPAGLEADGSRQARVGS (SEQ ID NO: 27); TGPAGLGLEADGSRQARVG (SEQ ID NO: 28); TGRQARVGLEADGSPAGLG (SEQ ID NO: 29); TGSRQARVGPAGLEADGS (SEQ ID NO: 30); and TGPAGLGSRQARVGLEADGS (SEQ ID NO: 31); GPAGLGLEPDGSRQARVG (SEQ ID NO: 104); GGSGGGGIEPDIGGSGGS (SEQ ID NO: 105); GGSGGGGLEADTGGSGGS (SEQ ID NO: 106); GSIEPDIGS (SEQ ID NO: 107); GSLEADTGS (SEQ ID NO: 108); ID NO: 108); GGSGGGGIEPDGGGSGGS (SEQ ID NO: 109); GGSGGGGIEPDVGGSGGS (SEQ ID NO: 110); GGSGGGGIEPDSGGSGGS (SEQ ID NO: 111); GGSGGGGIEPDTGGSGGS (SEQ ID NO: 112); GGGSLEPDGSGS (SEQ ID NO: 112); NO: 113); and GPAGLGLEADGSRQARVG (SEQ ID NO: 114), GGGEGGGGSGGSGGGGS (SEQ ID NO: 115); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 116); GGSGGGGLEAEGSGGGGS (SEQ ID NO: 117); GGSGGGGIEPDPGGSGGS (SEQ ID NO: 117); NO: 118); TGGGSGGGIEPDIGGSGGS (SEQ ID NO: 148).

[0314] 4. Antigen binding domain:

[0315] The multispecific polypeptide constructs of the present invention include at least one antigen binding domain, such as at least a first antigen binding domain and a second antigen binding domain. In some aspects, the antigen binding domain, or each antigen binding domain independently, is selected from an antibody or antigen binding fragment, a natural cognate binding partner, anticalin (engineered lipocalin), darpin, fynomer, centyrin (engineered fibronectin III domain), cysteine-knob domain, affilin, affibody, or an engineered CH3 domain. In some embodiments, the natural cognate binding partner comprises the extracellular domain of a natural cognate binding partner of a TAA, or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA.

[0316] In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain comprises one or more copies of an antibody or an antigen binding fragment thereof. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain comprises one or more copies of an antibody or an antigen binding fragment thereof selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain comprises one or more single domain antibody (sdAb) fragments, such as V H H, V NAR , modified V H or V K Domain. V H H can be generated from natural camelid heavy chain only antibodies, genetically modified rodents producing heavy chain only antibodies, or naive / synthetic camelid or humanized camelid single domain antibody libraries. NAR Heavy chain antibodies can be generated from cartilaginous fish only. Various methods have been performed to generate heterodimeric V H and V K The domains generate monomeric sdAbs, including surface engineering and selection of specific germline families. In some embodiments, the antigen binding domain of the multispecific polypeptide construct, or independently each antigen binding domain, such as the first antigen binding domain and / or the second antigen binding domain, contains VH and VL sequences assembled into a FAB or scFv. In some embodiments, the antigen binding domain of the multispecific polypeptide construct, or independently each antigen binding domain, such as the first antigen binding domain and / or the second antigen binding domain, contains a binding domain that is a single domain antibody (sdAb).

[0317] In some embodiments, the antigen binding domain, or independently each antigen binding domain, is or comprises the extracellular domain of a natural cognate binding partner of a TAA, or a binding fragment thereof, or a variant thereof that exhibits binding activity to a TAA.

[0318] In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to the same antigen. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to different antigens. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to the same tumor-associated antigen (TAA). In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to different TAAs. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to different epitopes on the same TAA. In some embodiments, the antigen binding domain, or independently each antigen binding domain, such as the first antigen binding domain and the second antigen binding domain, binds to the same epitope on the same TAA.

[0319] In some embodiments, the antigen binding domain that binds to a TAA, or each antigen binding domain independently, binds to a TAA monovalently, bivalently, trivalently, or tetravalently.

[0320] In some embodiments, the TAA is selected from the group consisting of: 1-92-LFA-3, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, april J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44 v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), Claudin-3, Claudin-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR 4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT=3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6,IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nacasterin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine phosphate 1, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4 , TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0321] In some embodiments, at least one antigen binding domain, or independently each antigen binding domain, binds to the tumor-associated antigen (TAA) folate receptor alpha (FRα). For example, the antigen binding domain comprises a binding domain that is a sdAb that binds FRα. Exemplary FRα-binding sdAbs are set forth in SEQ ID NOs: 120, 121, and 122.

[0322] In some embodiments, at least one antigen binding domain, or each antigen binding domain independently, binds to the tumor-associated antigen (TAA) cMET. For example, the antigen binding domain comprises a binding domain that is a sdAb that binds cMET. An exemplary cMET-binding sdAb is set forth in SEQ ID NO: 123 (U.S. Patent No. 9,346,884).

[0323] In some embodiments, at least one antigen-binding domain, or each antigen-binding domain independently, binds to the tumor-associated antigen (TAA) B7H3. For example, the antigen-binding domain comprises a binding domain that is a scFv that binds B7H3. An exemplary B7H3-binding scFv is set forth in SEQ ID NO: 124. In some embodiments, the antigen-binding domain is or comprises a Fab antibody fragment comprising a VH-CH1 (Fd) and a LC. An exemplary B7H3 Fd is set forth in SEQ ID NO: 127, and an exemplary B7H3 LC is set forth in SEQ ID NO: 128 (PCT Publication No. WO2017 / 030926).

[0324] In some embodiments, the antigen binding domain binds to the tumor-associated antigen (TAA) CD20. For example, the antigen binding domain comprises a binding domain that is a scFv that binds CD20. Exemplary CD20-binding scFvs are set forth in SEQ ID NOs: 125, 189, and 190 (U.S. Publication No. US2005 / 0123546).

[0325] In some embodiments, the antigen binding domain binds to the tumor-associated antigen (TAA) DLL3. For example, the antigen binding domain comprises a binding domain that is a scFv that binds DLL3. Exemplary DLL3-binding scFvs are set forth in SEQ ID NOs: 126 and 189 (U.S. Publication No. US2017 / 0037130). In some embodiments, the antigen binding domain is or comprises a Fab antibody fragment comprising an Fd and LC that binds DLL3. An exemplary DLL3 Fd is set forth in SEQ ID NO: 133, and an exemplary DLL3 LC is set forth in SEQ ID NO: 134 (U.S. Patent No. 8,044,178).

[0326] In some embodiments, the antigen binding domain binds to the tumor-associated antigen (TAA) 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 129 and an exemplary 5T4 LC is set forth in SEQ ID NO: 130. In some embodiments, the antibody binding domain comprises a VH-CH1 (Fd) or VL-CL as set forth in SEQ ID NOs: 167 and 168 (U.S. Pat. No. 8,044,178).

[0327] In some embodiments, the antigen binding domain binds to the tumor-associated antigen (TAA) gpNMB. In some embodiments, the antigen binding domain is or comprises a Fab fragment comprising an Fd and LC chain. An exemplary gpNMB Fd is set forth in SEQ ID NO: 131 and an exemplary gpNMB LC is set forth in SEQ ID NO: 132.

[0328] In some embodiments, the antigen binding domain is linked directly or indirectly to the Fc region and / or CD3 binding region via a linker. In some embodiments, the link is via a linker. In some embodiments, the linker is a connecting peptide (LP), which can include any flexible or rigid linker as shown in Section II.3, but peptides connecting one or more antigen binding domains are generally not cleavable linkers.

[0329] In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region. In some embodiments, the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the CD3 binding region and the second antigen-binding domain. In some embodiments, the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region and a second connecting peptide (LP2) between the CD3 binding region and the second antigen-binding domain. In some aspects, the multispecific polypeptide construct has the following structural configuration from N-terminus to C-terminus: first antigen-binding domain-LP1-Fc region-linker-CD3 binding region-LP2-second antigen-binding domain. In some embodiments, the two connecting peptides are different.

[0330] In some embodiments, LP1 or LP2 is independently a peptide of about 1 to 20 amino acids in length. In some embodiments, LP1 or LP2 is independently a peptide that is or comprises any Glu-Ser linker as shown in SEQ ID NOs: 10-13, 119, 135, 147, 149, or GGS.

[0331] III. Pharmaceutical Compositions

[0332] Provided herein are compositions of any of the provided multispecific polypeptide constructs. It will be understood that administration of the therapeutic entities according to the present disclosure should be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. A variety of suitable formulations are available for formulations known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pennsylvania (1975)), particularly Chapter 87 therein, Blaug, Seymour. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., Lipofectin TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the above mixtures may be suitable for use in the treatment and therapy according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated with the route of administration. For additional information on formulations, excipients, and carriers familiar to pharmaceutical chemists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2): 210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8): 967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations." PDA J Pharm Sci Technol. 52: 238-311 (1998) and references therein.

[0333] In some embodiments, the multispecific polypeptide constructs, coupled multispecific polypeptide constructs, and compositions thereof (collectively referred to herein as therapeutic agents and derivatives, fragments, analogs, and homologs thereof) can be incorporated into pharmaceutical compositions suitable for administration. Principles and considerations involved in preparing such compositions, as well as guidance for the selection of components, are provided in, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th ed. (Alfonso R. Gennaro et al., eds.), Mark Publishing Company, Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0334] Such compositions typically comprise a multispecific polypeptide construct or a conjugate thereof and a pharmaceutically acceptable carrier. If the multispecific polypeptide construct comprises an antibody fragment, the smallest fragment of the antibody that specifically binds to the target protein can be used. For example, based on the variable region sequence of an antibody, a peptide molecule can be designed that retains the antibody's ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0335] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and similar agents compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the industry, which is incorporated herein by reference. Suitable embodiments of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (e.g., fixed oils) may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or agents that are incompatible with the active compound, their use in therapeutic compositions is also contemplated.

[0336] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0337] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Embodiments of the route of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tension (e.g., sodium chloride or dextrose). The pH can be adjusted using an acid or base (e.g., hydrochloric acid or sodium hydroxide). Parenteral formulations can be encapsulated in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0338] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and its fluidity should be such that it has easy injection. It must be stable under manufacturing and storage conditions and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing (for example) water, ethanol, polyols (for example glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (for example lecithin), by maintaining the desired particle size (in the case of a dispersion) and by using a surfactant. Prevention of the effect of microorganisms can be achieved by various antibacterial and antifungal agents (for example, parahydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents (for example sugars, polyols (for example, mannitol, sorbitol) or sodium chloride) are suitably incorporated into the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0339] Sterile injectable solutions can be prepared by the following steps: the active compound of the desired amount is incorporated into a suitable solvent containing one or a combination (if necessary) of the ingredients listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, which can produce a powder consisting of the active ingredient and any desired additional ingredients (from its previously sterile-filtered solution). If the composition is freeze-dried, sterilization using this method can be performed before or after freeze-drying and reconstruction. The composition administered parenterally can be stored in a lyophilized form or in a solution. In addition, parenteral compositions are typically placed in a container with an infusion port, such as an intravenous solution bag or bottle with a stopper that can be pierced by a hypodermic needle.

[0340] In some embodiments, the pharmaceutical composition is administered to a subject via any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly applied to a wound site, applied to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by aerosolization, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intraspinally.

[0341] Oral compositions typically include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, sugar tablets, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and gargled and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of similar properties: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, carboxymethyl starch (Primogel), or corn starch; a lubricant such as magnesium stearate or fully hydrogenated vegetable oil (Sterotes); a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as mint, methyl salicylate, or orange flavor.

[0342] For administration by inhalation, the multispecific polypeptide constructs are delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant (eg, a gas such as carbon dioxide), or a nebulizer.

[0343] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the permeability barrier are used in the formulation. Such penetrants are generally known in the art and, for example, include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel, or cream as generally known in the art.

[0344] The compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0345] In one embodiment, therapeutic agents are prepared with carriers that protect the compound from rapid elimination from the body, such as sustained / controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0346] For example, the therapeutic agent can be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions prepared, for example, by coacervation techniques or surface polymerization, respectively.

[0347] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating, preservative, lubricant, flavoring, sweetener, colorant, solvent, buffer, chelating agent, or stabilizer. Examples of pharmaceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically acceptable binders include polyvinyl pyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Examples of pharmaceutically acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, zein, or gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinyl pyrrolidone, carboxymethylcellulose, or sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, or sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0348] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, and such matrices are in the form of formed articles, such as films or microcapsules. In some embodiments, the pharmaceutical composition further includes reagents for controlling or sustained release products, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads or liposomes. The example of sustained release matrix includes polyesters, hydrogels (such as poly (methacrylic acid-2-hydroxyethyl ester) or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time.

[0349] Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. Such materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0350] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of subjects.

[0351] Further provided are kits comprising the pharmaceutical compositions (or products) described herein. Such pharmaceutical compositions can be enclosed in a container, package, or dispenser along with instructions for administration. The kits described herein may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for practicing any of the methods described herein.

[0352] The formulation may also contain more than one multispecific polypeptide construct as needed for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other. In some embodiments, or in addition, the composition may include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for their intended purpose.

[0353] In some embodiments, the dosage of the pharmaceutical composition is a single dose or repeated dose. In some embodiments, the dosage is once a day, twice a day, three times a day or four times a day or more administered to the subject. In some embodiments, about 1 or more (e.g., about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more or about 7 or more) dosages are administered within a week. In some embodiments, multiple dosages are administered over a period of several days, weeks, months or years. In some embodiments, the course of treatment is about 1 or more dosages (e.g., about 2 or more dosages, about 3 or more dosages, about 4 or more dosages, about 5 or more dosages, about 7 or more dosages, about 10 or more dosages, about 15 or more dosages, about 25 or more dosages, about 40 or more dosages, about 50 or more dosages or about 100 or more dosages).

[0354] In some embodiments, a pharmaceutical composition is administered to a subject. Typically, the dosage and route of administration of the pharmaceutical composition are determined according to the size and condition of the subject, according to standard medical practice. For example, a therapeutically effective dose can be initially estimated in a cell culture assay or in an animal model (e.g., mouse, rat, rabbit, dog, pig, or monkey). Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine the available dosage and route of administration in humans. The precise dosage will be determined based on factors related to the subject in need of treatment. The dosage is adjusted and administered to provide a sufficient amount of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's general health, the subject's age, weight, and sex, administration time and frequency, drug combination, reaction sensitivity, and response to therapy. A person skilled in the art of medicine can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's signs of the disease and adjusting the treatment accordingly.

[0355] IV. Methods of Use and Therapeutic Administration

[0356] Also provided are methods of using the multispecific polypeptide constructs and uses thereof. Such methods and uses include therapeutic methods and uses, for example, involving administering a molecule or a composition comprising the same to a subject having a disease, condition, or disorder, such as a tumor or cancer. In some embodiments, the molecule and / or composition is administered in an amount effective to effectively treat the disease or condition. Uses include use of the multispecific polypeptide constructs in such methods and treatments, and use in the preparation of a medicament for performing such therapeutic methods. In some embodiments, the methods are performed by administering a multispecific polypeptide construct, or a composition comprising the same, to a subject having or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease, condition, or disorder in the subject.

[0357] In one embodiment, the multispecific polypeptide constructs of the present disclosure can be used as therapeutic agents. Such agents will generally be used to diagnose, prognose, monitor, treat, alleviate, and / or prevent a disease or condition in a subject. The treatment regimen is implemented by identifying a subject (e.g., a human patient or other mammal) suffering from (or at risk of developing) a condition using standard methods. The multispecific polypeptide construct is administered to the subject. The multispecific polypeptide construct is administered to the subject and will generally have an effect due to its binding to the target.

[0358] In some embodiments, provided herein are methods for modulating an immune response in a subject by administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the methods for modulating an immune response increase or enhance the immune response of a subject. For example, the increased or enhanced response can be an increase in cell-mediated immunity. In some embodiments, the methods increase T cell activity, such as cytolytic T cell (CTL) activity. In some embodiments, the immune response that is modulated (e.g., increased) is directed against a tumor or cancer.

[0359] Administration of a multispecific polypeptide construct can activate innate immune cells via FcγR engagement by the Fc-region of the multispecific polypeptide construct. Administration of a multispecific polypeptide construct can excite, stimulate, activate, and / or amplify innate immune cell effector functions, including ADCC, cytokine release, degranulation, and / or ADCP. Administration of a multispecific polypeptide construct can activate T cells once the linker connecting the first and second components is cleaved by a protease, thereby allowing the anti-CD3 binding moiety to bind to CD3ε on the T cell. Administration of a multispecific polypeptide construct can excite, stimulate, activate, and / or amplify CD3-mediated T cell activation, cytotoxicity, cytokine release, and / or proliferation.

[0360] In some embodiments, the methods provided are used to treat a disease or condition of a subject by administering a therapeutically effective amount of any of the provided multispecific conjugates or pharmaceutical compositions. In some embodiments, the disease or condition is a tumor or cancer. Typically, alleviating or treating a disease or condition involves alleviating one or more symptoms or medical problems associated with the disease or condition. For example, in the case of cancer, a therapeutically effective amount of a drug can accomplish one or a combination of the following: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., reducing to a certain extent / or stopping) cancer cell infiltration into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth to a certain extent; and / or alleviating one or more symptoms associated with cancer to a certain extent. In some embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or condition in a subject (e.g., a human or other mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), a farm animal, a working animal, or a zoo animal). The terms subject and patient are used interchangeably herein.

[0361] In some embodiments, the pharmaceutical composition can be used to inhibit the growth of mammalian cancer cells (e.g., human cancer cells). The method of treating cancer may include administering an effective amount of any of the pharmaceutical compositions described herein to a subject suffering from cancer. An effective amount of the pharmaceutical composition can be administered to inhibit, prevent, or reverse the progression of cancer. Human cancer cells can be treated in vivo or ex vivo. In ex vivo treatment of human patients, tissue or fluid containing cancer cells is treated in vitro, and then the tissue or fluid is redirected back into the patient. In some embodiments, in some embodiments, cancer is treated in a human patient by administering a therapeutic composition to the patient.

[0362] Non-limiting examples of diseases include: all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck cancer, and pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, ischemia, etc. For example, indications would include leukemia (including T-cell acute lymphoblastic leukemia (T-ALL)), lymphoblastic diseases (including multiple myeloma), and solid tumors (including lung tumors, colorectal tumors, prostate tumors, pancreatic tumors, and breast tumors (including triple-negative breast cancer)). For example, indications include bone disease or cancer metastasis that is unrelated to the source of the primary tumor; breast cancer, including (by way of non-limiting embodiment) ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as, by way of non-limiting embodiment, non-small cell lung cancer; multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as, by way of non-limiting embodiment, renal cell carcinoma; and / or skin cancer, such as, by way of non-limiting embodiment, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0363] A therapeutically effective amount of a multispecific polypeptide construct of the present disclosure generally relates to the amount required to achieve the therapeutic goal. As described above, this can be a binding interaction between the multispecific polypeptide construct and its target antigen, which in some cases excites, stimulates, activates and / or increases FcγR-mediated innate immune cell activation or CD3-mediated T cell activation. The required administration amount will further depend on the binding affinity of the multispecific polypeptide construct for its specific antigen, and also on the rate at which the administered multispecific polypeptide construct is depleted of its free volume from other subjects to which it is administered. A common range of therapeutically effective doses of a multispecific polypeptide construct can be (by way of non-limiting embodiment) from about 0.01 μg / kg body weight to about 10 mg / kg body weight. In some embodiments, a therapeutically effective dose of a multispecific polypeptide construct of the present disclosure can be (by way of non-limiting embodiment) from about 0.01 mg / kg body weight to about 5-10 mg / kg body weight. Common dosing frequencies can range from, for example, twice daily to once a week.

[0364] The effectiveness of the treatment is determined in conjunction with any known method for diagnosing or treating a particular condition. Methods for screening multispecific polypeptide constructs with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immune-mediated techniques known in the art. A variety of methods are known for determining whether administration of a provided multispecific polypeptide construct is sufficient to modulate immune activity by: inducing, generating, or activating immune cells that mediate or are capable of mediating a protective immune response; altering the physical or functional properties of immune cells; or a combination of such effects. Examples of measurements of modulation of immune activity include, but are not limited to, examining the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measuring the functional capacity of immune cells, including the ability to proliferate or divide in response to signals or their resistance to such signals (e.g., using T-cell proliferation assays and peptide scanning assays based on 3H-thymidine incorporation following stimulation with anti-CD3 antibodies, anti-T cell receptor antibodies, anti-CD28 antibodies, calcium ionophores, PMA (phorbol 12-myristate 13-acetate) antigen-presenting cells loaded with peptide or protein antigens; B cell proliferation assays); measuring the ability to kill or lyse other cells (e.g., cytotoxic T cell assays); measuring cytokines, chemokines, cell surface molecules, antibodies, and other cellular products (e.g., by flow cytometry, enzyme-linked immunosorbent assays, Western blots, protein microarrays, immunoprecipitation assays); measuring the activity of immune cells or activation of signaling pathways within immune cells. measurement of cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assay, gel electrophoresis to measure DNA ladders, histology; assays for caspases using fluorescent probes, Western blot analysis for caspase substrates); measurement of genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarrays, protein microarrays, two-dimensional gel electrophoresis, Western blot analysis, enzyme-linked immunosorbent assay, flow cytometry); measurement of clinical symptoms or outcomes, such as by measuring relapse rate or disease severity, such as improvement (clinical scores, need for additional therapy, functional status, imaging studies) of autoimmune, neurodegenerative, and other diseases involving self-proteins or self-peptides.

[0365] Multispecific polypeptide constructs can also be used in various diagnostic and prophylactic formulations. In one embodiment, a multispecific polypeptide construct is administered to a patient at risk of developing one or more of the conditions mentioned above. The predisposition of a patient or organ to one or more of the conditions can be determined using genotypic, serological, or biochemical markers.

[0366] In another embodiment of the present invention, a multispecific polypeptide construct is administered to a human subject diagnosed with a clinical indication associated with one or more of the above-mentioned conditions. At the time of diagnosis, the multispecific polypeptide construct is administered to alleviate or reverse the effects of the clinical indication.

[0367] Combination therapy

[0368] In some embodiments, a multispecific polypeptide construct, a coupled multispecific polypeptide construct, and compositions thereof (collectively referred to herein as a therapeutic agent) is administered together with one or more additional agents or a combination of additional agents. Suitable additional agents include current medical and / or surgical therapies for the intended application. For example, a therapeutic agent can be used together with an additional chemotherapeutic agent or an anti-tumor agent. For example, the therapeutic agent and the additional agent are formulated into a single therapeutic composition, and the therapeutic agent and the additional agent are administered simultaneously. In some embodiments, the therapeutic agent and the additional agent are separated from each other, for example, each is formulated into a separate therapeutic composition, and the therapeutic agent and the additional agent are administered simultaneously, or the therapeutic agent and the additional agent are administered at different times during the treatment regimen. For example, the therapeutic agent is administered before the additional agent, the therapeutic agent is administered after the additional agent, or the therapeutic agent and the additional agent are administered in an alternating manner. As described herein, the therapeutic agent and the additional agent are administered in a single dose or multiple doses. In some embodiments, the additional agent is coupled or otherwise attached to the therapeutic agent. Suitable additional agents are selected based on the purpose of the intended application (i.e., killing, preventing cell proliferation, hormone therapy, or gene therapy). Such agents may include, but are not limited to, for example, pharmaceutical agents, toxins, fragments of toxins, alkylating agents, enzymes, antibiotics, antimetabolites, antiproliferative agents, hormones, neurotransmitters, DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, diagnostic agents, radiopaque dyes, radioisotopes, fluorescent probe compounds, magnetic labels, nanoparticles, labeled compounds, lectins, compounds that alter cell membrane permeability, photochemical compounds, small molecules, liposomes, micelles, gene therapy vectors, viral vectors, etc. Finally, combinations of agents or combinations of different classes of agents may be used.

[0369] In one embodiment, the multispecific polypeptide construct is administered in combination therapy, i.e., in combination with other agents (e.g., therapeutic agents) useful for treating pathological conditions or disorders (e.g., autoimmune disorders and inflammatory diseases). The term "combination" in this context means administering such agents substantially simultaneously, concurrently, or sequentially. If administered sequentially, the first of the two compounds should still be detectable at an effective concentration at the treatment site at the start of administration of the second compound.

[0370] For example, combination therapies can include co-formulation and / or co-administration of one or more multispecific polypeptide constructs of the present disclosure with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in more detail below). In addition, one or more multispecific polypeptide constructs described herein can be used in combination with two or more therapeutic agents described herein. Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0371] In other embodiments, one or more multispecific polypeptide constructs of the present disclosure are co-formulated and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. Non-limiting examples of drugs or inhibitors that can be used in combination with the antibodies described herein include, but are not limited to, one or more of the following: nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids, such as prednisone; ne); cytokine suppressive anti-inflammatory drugs (CSAIDs); inhibitors of nucleotide biosynthesis, for example, inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamate); and inhibitors of pyrimidine biosynthesis, for example, dihydroorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the present disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate).

[0372] Examples of additional inhibitors include one or more of the following: corticosteroids (oral, inhaled, and local injection); immunosuppressants, such as cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, such as sirolimus (rapamycin-RAPAMUNE TM or rapamycin derivatives, such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives, such as CCI-779); agents that interfere with signaling by proinflammatory cytokines, such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, such as celecoxib, rofecoxib and variants thereof; phosphodiesterase inhibitors, such as R973401 (phosphodiesterase type IV inhibitor); phospholipase inhibitors, such as inhibitors of cytoplasmic phospholipase 2 (cPLA2) (e.g., trifluoromethylketone analogs); inhibitors of vascular endothelial growth factor or growth factor receptors, such as VEGF inhibitors and / or VEGF-R inhibitors; and inhibitors of angiogenesis. Suitable therapeutic agents for use in combination with the disclosed antibodies are immunosuppressants, such as cyclosporine, tacrolimus (FK-506); mTOR inhibitors, such as sirolimus (rapamycin) or rapamycin derivatives, such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives, such as CCI-779); COX2 inhibitors, such as celecoxib and its variants; and phospholipase inhibitors, such as inhibitors of cytosolic phospholipase 2 (cPLA2), such as trifluoromethylketone analogs. Additional examples of therapeutic agents that can be combined with the multispecific polypeptide constructs include one or more of the following: 6-mercaptopurine (6-MP); azathioprine sulfasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine Penicillamine; gold thiomalate (intramuscular and oral); azathioprine; colchicine; beta-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeterol); xanthines (theophylline, amine theophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0373] V. Exemplary Embodiments

[0374] The embodiments provided are:

[0375] 1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein:

[0376] The first component and the second component are coupled by a linker, wherein the Fc region is located N-terminal to the CD3 binding region; and

[0377] One or both of the first component and the second component comprises an antigen binding domain that binds a tumor associated antigen (TAA).

[0378] 2. The multispecific polypeptide construct of embodiment 1, wherein the CD3 binding region binds to CD3 (CD3ε).

[0379] 3. The multispecific construct of embodiment 1 or embodiment 2, wherein the antigen binding domain is located amino-terminally relative to the Fc region and / or carboxy-terminally relative to the CD3 binding region of the multispecific polypeptide construct.

[0380] 4. The multispecific polypeptide construct of any one of embodiments 1 to 3, wherein the first component comprises a first antigen binding domain and the second component comprises a second antigen binding domain, wherein each of such antigen binding domains binds to a tumor associated antigen (TAA).

[0381] 5. The multispecific polypeptide construct of embodiment 4, wherein the first antigen binding domain is located amino-terminal to the Fc region of the multispecific construct and the second antigen binding domain is located carboxyl-terminal to the CD3 binding region of the multispecific construct.

[0382] 6. A multispecific polypeptide construct, wherein the multispecific construct comprises, from N-terminus to C-terminus:

[0383] a first antigen binding domain that binds to a tumor-associated antigen (TAA);

[0384] Immunoglobulin Fc region;

[0385] connector;

[0386] a CD3 binding region that binds CD3 (CD3ε); and

[0387] The second antigen binding domain binds a tumor associated antigen (TAA).

[0388] 7. A multispecific polypeptide construct, wherein the multispecific construct comprises, from N-terminus to C-terminus:

[0389] Immunoglobulin Fc region;

[0390] connector;

[0391] a CD3 binding region that binds CD3 (CD3ε); and

[0392] Antigen binding domain that binds to a tumor-associated antigen (TAA).

[0393] 8. A multispecific polypeptide construct, wherein the multispecific construct comprises, from N-terminus to C-terminus:

[0394] an antigen binding domain that binds to a tumor-associated antigen (TAA);

[0395] Immunoglobulin Fc region;

[0396] connectors; and

[0397] Binds the CD3 binding region of CD3 (CD3ε).

[0398] 9. The multispecific polypeptide construct of any one of embodiments 1 to 8, wherein the Fc region is a homodimeric Fc region.

[0399] 10. The multispecific polypeptide construct of any one of embodiments 1 to 9, wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof.

[0400] 11. The multispecific polypeptide construct of any one of embodiments 1 to 10, wherein the Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0401] 12. The multispecific polypeptide construct of any one of embodiments 1 to 10, wherein the Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2;

[0402] The Fc region comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4; or

[0403] The Fc region comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.

[0404] 13. The multispecific polypeptide construct of any one of embodiments 1 to 6, 9 and 12, wherein the Fc region is a heterodimeric Fc region.

[0405] 14. A multispecific polypeptide construct as described in embodiment 13, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to induce heterodimerization compared to the polypeptide of the homodimeric Fc region, optionally compared to the Fc polypeptide shown in SEQ ID NO: 1 or an immunologically active fragment thereof.

[0406] 15. The multispecific polypeptide construct of embodiment 14, wherein each of such Fc polypeptides of the heterodimeric Fc independently comprises at least one amino acid modification.

[0407] 16. The multispecific polypeptide construct of embodiment 15, wherein each of the Fc polypeptides of the heterodimeric Fc comprises a knob-to-hole structure modification or comprises a charge mutation to increase the electrostatic complementarity of the polypeptides.

[0408] 17. The multispecific polypeptide construct of embodiment 16, wherein the amino acid modification is a knob-to-hole structure modification.

[0409] 18. The multispecific fusion polypeptide of any one of embodiments 13 to 17, wherein the first Fc polypeptide of the heterodimeric Fc comprises a modification selected from Thr3 66Ser, Leu3 68A1a, Tyr407Va1, and a combination thereof, and the second Fc polypeptide of the heterodimeric Fc comprises the modification T366W.

[0410] 19. The multi-specific fusion polypeptide of embodiment 18, wherein the first Fc polypeptide and the second Fc polypeptide further comprise a modification of a non-cysteine ​​residue to a cysteine ​​residue, wherein the modification of the first polypeptide is at one of positions Ser354 and Y349 and the modification of the second Fc polypeptide is at the other of positions Ser354 and Y349.

[0411] 20. The multispecific polypeptide construct of embodiment 16, wherein the amino acid modification is a charge mutation to increase electrostatic complementarity of such polypeptides.

[0412] 21. The multispecific polypeptide construct of any one of embodiments 13 to 16 and 20, wherein the first Fc polypeptide and / or the second Fc polypeptide or each of the first Fc polypeptide and the second Fc polypeptide comprises a modification of a complementary position, wherein the modification is a replacement with an amino acid having a charge opposite to that of the complementary amino acid of the other polypeptide.

[0413] 22. The multispecific polypeptide construct of any one of embodiments 14 to 21, wherein one of the first Fc polypeptide or the second Fc polypeptide of the heterodimeric Fc further comprises modification of residue Ile253.

[0414] 23. The multispecific polypeptide construct of embodiment 22, wherein the modification is Ile253Arg.

[0415] 24. The multispecific polypeptide construct of any one of embodiments 14 to 23, wherein one of the first Fc polypeptide or the second Fc polypeptide of the heterodimeric Fc further comprises modification of residue His435.

[0416] 25. The multispecific polypeptide construct of embodiment 24, wherein the modification is His435Arg.

[0417] 26. The multispecific polypeptide construct of any one of embodiments 1 to 25, wherein the Fc region comprises a polypeptide without Lys447.

[0418] 27. The multispecific polypeptide construct of any one of embodiments 1 to 26, wherein the Fc region comprises a polypeptide comprising at least one modification to enhance FcRn binding.

[0419] 28. The multi-specific fusion polypeptide of embodiment 27, wherein the modification is at a position selected from the group consisting of Met252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

[0420] 29. The multi-specific fusion polypeptide of embodiment 28, wherein the modification is at a position selected from the group consisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinations thereof.

[0421] 30. The multi-specific fusion polypeptide of embodiment 28, wherein the modifications are at position Met252 and position Met428.

[0422] 31. The multi-specific fusion polypeptide of embodiment 30, wherein the modifications are Met252Y and Met428L.

[0423] 32. The multi-specific fusion polypeptide of embodiment 30, wherein the modifications are Met252Y and Met428V.

[0424] 33. The multispecific polypeptide construct of any one of embodiments 13 to 32, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 82, 86, 94 or 96, and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 83, 87, 90, 92, 98 or 100.

[0425] 34. The multispecific polypeptide construct of any one of embodiments 1 to 33, wherein the Fc region comprises a polypeptide comprising at least one amino acid modification that reduces effector function and / or reduces binding to an effector molecule selected from an Fcγ receptor or C1q.

[0426] 35. The multispecific polypeptide construct of embodiment 34, wherein the one or more amino acid modifications are deletions of one or more of Glu233, Leu234 or Leu235.

[0427] 36. The multispecific polypeptide construct of any one of embodiments 13 to 32, 34 and 35, wherein the first polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 84, 88, 95 or 97 and the second polypeptide of the heterodimeric Fc comprises the amino acid sequence shown in any one of SEQ ID NO: 85, 89, 91, 93, 99 or 101.

[0428] 37. The multispecific polypeptide construct of any one of embodiments 1 to 32, wherein the Fc region comprises a polypeptide comprising at least one modification to enhance FcγR binding.

[0429] 38. The multispecific polypeptide construct of embodiment 37, wherein the modification is modification of Ser239 or Ile332.

[0430] 39. The multispecific polypeptide construct of any one of embodiments 1 to 32 and 37, wherein the glycosylation of the Fc region is modified to enhance FcγR binding compared to an unmodified Fc region.

[0431] 40. The multispecific polypeptide construct of embodiment 39, wherein the Fc region has no or reduced fucose content.

[0432] 41. The multispecific polypeptide construct of any one of embodiments 1 to 40, wherein the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment.

[0433] 42. The multispecific polypeptide construct of embodiment 41, wherein the anti-CD3 antibody or antigen-binding fragment comprises a variable heavy chain region (VH) and a variable light chain region (VL).

[0434] 43. The multispecific polypeptide construct of any one of embodiments 1 to 42, wherein the CD3 binding region is monovalent.

[0435] 44. The multispecific polypeptide construct of any one of embodiments 41 to 43, wherein the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody, optionally not a single-chain variable fragment (scFv).

[0436] 45. The multispecific polypeptide construct of embodiment 42 or embodiment 44, wherein the Fc is a heterodimeric Fc, and the VH and the VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc.

[0437] 46. ​​The multispecific polypeptide construct of any one of embodiments 1 to 45, wherein the CD3 binding region is unable or substantially unable to bind or engage CD3 unless at least one of the antigen binding domains is bound to its TAA.

[0438] 47. The multispecific polypeptide construct of any one of embodiments 1 to 46, wherein the CD3 binding region is unable or substantially unable to bind or engage CD3 unless at least two of the antigen binding domains are bound to their TAAs.

[0439] 48. The multispecific polypeptide construct of any one of embodiments 1 to 47, wherein the linker is a polypeptide linker.

[0440] 49. The multispecific polypeptide construct of embodiment 48, wherein the linker is a polypeptide of up to 25 amino acids in length.

[0441] 50. The multispecific polypeptide construct of embodiment 48 or embodiment 49, wherein the linker is or is about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 or a polypeptide of 20 to 24 amino acids.

[0442] 51. The multispecific polypeptide construct of any one of embodiments 48 to 50, wherein the linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0443] 52. The multispecific polypeptide construct of any one of embodiments 1 to 51, wherein the linker is a cleavable linker.

[0444] 53. A multispecific polypeptide construct comprising a first component comprising a heterodimeric Fc region and a second component comprising an anti-CD3 antibody or antigen-binding fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL), wherein:

[0445] The VH and VL constituting the anti-CD3 antibody or antigen-binding fragment are linked to opposite polypeptides of the heterodimeric Fc;

[0446] The first component and the second component are coupled by a cleavable linker, wherein the heterodimeric Fc region is located at the N-terminus of the anti-CD3 antibody; and

[0447] One or both of the first component and the second component comprises an antigen binding domain that binds a tumor associated antigen (TAA).

[0448] 54. The multispecific polypeptide construct of embodiment 52 or embodiment 53, wherein the binding of the CD3 binding region to CD3 is significantly reduced when the multispecific polypeptide construct is in an uncleaved state compared to a lysed state.

[0449] 55. The multispecific polypeptide of any one of embodiments 52 to 54, wherein in the cleaved state, the first component and the second component are not linked.

[0450] 56. The multispecific polypeptide construct of any one of embodiments 52 to 55, wherein the cleavable linker is a polypeptide that serves as a substrate for a protease.

[0451] 57. The multispecific polypeptide construct of embodiment 56, wherein the protease is produced by an immune effector cell, by a tumor, or by cells present in the tumor microenvironment.

[0452] 58. The multispecific polypeptide construct of embodiment 57, wherein the protease is produced by an immune effector cell, and the immune effector cell is an activated T cell, a natural killer (NK) cell, or an NK T cell.

[0453] 59. The multispecific polypeptide construct of any one of embodiments 56 to 58, wherein the protease is selected from the group consisting of an interstitial protease, a matrix metalloproteinase (MMP), granzyme B, and combinations thereof.

[0454] 60. The multispecific polypeptide construct of embodiment 59, wherein the protease is granzyme B.

[0455] 61. The multi-specific polypeptide construct of any one of embodiments 52 to 60, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓P1' (SEQ ID NO: 150), wherein P4 is amino acid I, L, Y, M, F, V, or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P, A, V, G, S, or T; P1 is amino acid D or E; and P1' is amino acid I, L, Y, M, F, V, T, S, G, or A.

[0456] 62. The multispecific polypeptide construct of any one of embodiments 52 to 61, wherein the cleavable linker comprises an amino acid sequence of the general formula P4 P3 P2 P1 ↓P1' (SEQ ID NO: 151), wherein P4 is amino acid I or L; P3 is amino acid E; P2 is amino acid P or A; P1 is amino acid D; and P1' is amino acid I, V, T, S, or G.

[0457] 63. The multispecific polypeptide construct of any one of embodiments 52 to 62, wherein the cleavable linker comprises the amino acid sequence IEPDI (SEQ ID NO: 136), LEPDG (SEQ ID NO: 152), LEADT (SEQ ID NO: 137), IEPDG (SEQ ID NO: 138), IEPDV (SEQ ID NO: 139), IEPDS (SEQ ID NO: 140), IEPDT (SEQ ID NO: 141), or LEADG (SEQ ID NO: 153).

[0458] 64. The multispecific polypeptide construct of any one of embodiments 52 to 63, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 105-112, 136-141, 148, 150-153.

[0459] 65. The multispecific polypeptide construct of embodiment 59, wherein the protease is an interstitial protease.

[0460] 66. The multispecific polypeptide construct of any one of embodiments 52 to 65, wherein:

[0461] The cleavable linker comprises the sequence P1QAR↓(A / V) (SEQ ID NO: 154), wherein P1 is any amino acid; or

[0462] The cleavable linker comprises the sequence RQAR(A / V) (SEQ ID NO: 155).

[0463] 67. The multispecific polypeptide construct of any one of embodiments 52 to 66, wherein the cleavable linker comprises the sequence RQARV (SEQ ID NO: 156).

[0464] 68. The multispecific polypeptide construct of any one of embodiments 52 to 67, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 154-156.

[0465] 69. The multispecific polypeptide construct of embodiment 59, wherein the protease is a MMP.

[0466] 70. The multispecific polypeptide construct of embodiment 69, wherein the MMP is MMP-2.

[0467] 71. The multispecific polypeptide construct of any one of embodiments 52 to 70, wherein the cleavable linker comprises the formula P3 P2 P1 ↓P1' (SEQ ID NO: 157), wherein P3 is P, V, or A; P2 is Q or D; P1 is A or N; and P1' is L, I, or M.

[0468] 72. The multispecific polypeptide construct of any one of embodiments 52 to 71, wherein the cleavable linker comprises the formula P3 P2 P1 ↓P1′ (SEQ ID NO: 158), wherein P3 is P; P2 is Q or D; P1 is A or N; and P1′ is L or I.

[0469] 73. The multispecific polypeptide construct of any one of embodiments 52 to 72, wherein the cleavable linker comprises the sequence PAGL (SEQ ID NO: 24).

[0470] 74. The multispecific polypeptide construct of any one of embodiments 52 to 73, wherein the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-31, 104-114, 117-118, 136-144, 148, 150-158.

[0471] 75. The multispecific polypeptide construct of any one of embodiments 45 to 74, wherein the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising the first Fc polypeptide of the heterodimeric Fc region, the linker, and the VH domain of the anti-CD3 antibody or antigen-binding fragment; and (ii) a second polypeptide comprising the second Fc polypeptide of the heterodimeric Fc region, the linker, and the VL domain of the anti-CD3 antibody or antigen-binding fragment, wherein one or both of the first polypeptide and the second polypeptide comprises at least one antigen binding domain that binds to a tumor associated antigen (TAA).

[0472] 76. The multispecific polypeptide construct of any one of embodiments 1 to 75, wherein one or more TAA-binding antigen binding domains bind monovalently, bivalently, trivalently, or tetravalently to the TAA.

[0473] 77. The multispecific polypeptide construct of embodiment 75, wherein only one of the first polypeptide or the second polypeptide comprises the at least one antigen binding domain that binds to TAA.

[0474] 78. The multispecific polypeptide construct of embodiment 75 or embodiment 77, wherein the at least one antigen binding domain is located at the amino terminus relative to the Fc region and / or at the carboxyl terminus relative to the CD3 binding region of one of the first polypeptide or the second polypeptide of the multispecific polypeptide construct.

[0475] 79. The multispecific polypeptide construct of embodiment 75 or embodiment 77, wherein the at least one antigen binding domain is located amino-terminal to the Fc region of the multispecific construct and the second antigen binding domain is located carboxyl-terminal to the CD3 binding region of the multispecific construct.

[0476] 80. The multispecific polypeptide construct of any one of embodiments 1 to 79, wherein the antigen binding domain or independently each of such antigen binding domains comprises an extracellular domain or binding fragment thereof of a natural cognate binding partner of the TAA or a variant thereof that exhibits binding activity to the TAA.

[0477] 81. The multispecific polypeptide construct of any one of embodiments 1 to 79, wherein the antigen binding domain or each of such antigen binding domains independently is an antibody or antigen binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0478] 82. The multispecific polypeptide construct of embodiment 81, wherein the antibody or antigen-binding fragment thereof is a Fv, scFv, Fab, a single domain antibody (sdAb), a V NAR or V H H.

[0479] 83. The multispecific polypeptide construct of embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment is an sdAb.

[0480] 84. The multispecific polypeptide construct of embodiment 83, wherein the sdAb is a human or humanized sdAb.

[0481] 85. A multispecific polypeptide construct as in embodiment 83 or embodiment 84, wherein the sdAb is V H H, V NA R , an engineered VH domain or an engineered VK domain.

[0482] 86. The multispecific polypeptide construct of embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment thereof is a scFv.

[0483] 87. The multispecific polypeptide construct of embodiment 81 or embodiment 82, wherein the antibody or antigen-binding fragment thereof is a Fab.

[0484] 88. The multispecific polypeptide construct of embodiment 87, wherein the multispecific polypeptide construct comprises:

[0485] (i) a first polypeptide comprising the first Fc polypeptide of the heterodimeric Fc region, the linker, and the VH domain of the anti-CD3 antibody or antigen-binding fragment;

[0486] (ii) a second polypeptide comprising the second Fc polypeptide of the heterodimeric Fc region, the linker and the VL domain of the anti-CD3 antibody or antigen-binding fragment, and

[0487] (iii) a third polypeptide comprising VH-CH1(Fd) or VL-CL of a Fab antibody fragment that binds to a tumor-associated antigen, wherein the first polypeptide and / or the second polypeptide further comprises the other of the VH-CH1(Fd) or the VL-CL of the Fab antibody fragment.

[0488] 89. The multispecific polypeptide construct of embodiment 88, wherein only one of the first polypeptide or the second polypeptide comprises the other of the VH-CH 1 (Fd) or the VL-CL of the Fab antibody fragment.

[0489] 90. The multispecific polypeptide construct of embodiment 89, wherein both the first polypeptide and the second polypeptide comprise the other of the VH-CH1 (Fd) or the VL-CL of the Fab antibody fragment.

[0490] 91. The multispecific polypeptide construct of embodiment 89 or embodiment 90, wherein the other of the VH-CH1 (Fd) or the VL-CL of the Fab antibody fragment is located at the amino terminus of one of the first polypeptide or the second polypeptide of the multispecific polypeptide construct relative to the Fc region and / or is located at the carboxyl terminus relative to the CD3 binding region.

[0491] 92. The multispecific polypeptide construct of any one of embodiments 89 to 91, wherein the other of the VH-CH1 (Fd) or the VL-CL of the Fab antibody fragment is located amino-terminally to the Fc region relative to the first polypeptide or the second polypeptide and carboxyl-terminally to the CD3 binding region relative to the other of the first polypeptide or the second polypeptide.

[0492] 93. The multispecific polypeptide construct of any one of embodiments 1 to 92, wherein the antigen binding domain, or each of such antigen binding domains independently, binds to a tumor antigen selected from the group consisting of: 1-92-LFA-3, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, april J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9 (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25 、CD27、CD28、CD30、CD33、CD38、CD40、CD40L、CD41、CD44、CD44v6、CD47、CD51、CD52、CD56、CD64、CD70、CD71、CD74、CD80、CD81、CD86、CD95、CD117、CD123、CD125、CD132、(IL-2RG)、CD133、CD137、CD138、CD166、CD172A、CD248、CDH6、CEACAM5(CEA)、CEACAM6(NCA-90)、Claunectin-3、Claunectin-4、cMet、Collagen, Cripto, C SFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nacastroin, Notch receptor, Notch1, Notch 2, Notch 3, Notch4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine phosphate 1, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0493] 94. The multispecific polypeptide construct of any one of embodiments 1 to 93, wherein the multispecific antigen binding domain comprises at least a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain and the second antigen binding domain bind to the same TAA.

[0494] 95. The multispecific polypeptide construct of embodiment 94, wherein the first antigen binding domain and the second antigen binding domain bind to different epitopes on the same TAA.

[0495] 96. The multispecific polypeptide construct of embodiment 94, wherein the first antigen binding domain and the second antigen binding domain bind to the same epitope on the same TAA.

[0496] 97. The multispecific polypeptide construct of any one of embodiments 1 to 96, wherein the multispecific antigen binding domain comprises at least a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds to a different TAA than the second antigen binding domain.

[0497] 98. The multispecific polypeptide construct of any one of embodiments 5 to 97, wherein the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen binding domain and the Fc region.

[0498] 99. The multispecific polypeptide construct of any one of embodiments 5 to 98, wherein the multispecific polypeptide construct comprises a second connecting peptide (LP2) between the CD3 binding region and the second antigen binding domain.

[0499] 100. The multispecific polypeptide construct of any one of embodiments 5 to 99, wherein the multispecific polypeptide construct comprises a first connecting peptide (LP1) between the first antigen-binding domain and the Fc region and a second connecting peptide (LP2) between the CD3 binding region and the second antigen-binding domain, and wherein the multispecific polypeptide construct has the following structural configuration from N-terminus to C-terminus: first antigen-binding domain-LP1-Fc region-linker-CD3 binding region-LP2-second antigen-binding domain.

[0500] 101. The multispecific polypeptide construct of embodiment 100, wherein the linker is a cleavable linker.

[0501] 102. The multispecific polypeptide construct of embodiment 100 and embodiment 101, wherein the two connecting peptides are different from each other.

[0502] 103. The multispecific polypeptide construct of any one of embodiments 98 to 102, wherein LP1 or LP2 is independently a peptide of about 1 to 20 amino acids in length.

[0503] 104. The multispecific polypeptide of embodiment 103, wherein LP1 or LP2 independently comprises a peptide that is or comprises any Gly-Ser linker as shown in SEQ ID NO: 10-13, 119, 135, 147, 149 or GGS.

[0504] 105. The multispecific polypeptide construct of any one of embodiments 41 to 104, wherein the anti-CD3 antibody or antigen-binding fragment is a Fv antibody fragment.

[0505] 106. The multispecific polypeptide construct of embodiment 105, wherein the Fv antibody fragment comprises a disulfide-stabilized anti-CD3 binding Fv fragment (dsFv).

[0506] 107. The multispecific polypeptide construct of any one of embodiments 41 to 106, wherein the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence of TYAMN (SEQ ID NO: 16); a VH CD2 comprising the amino acid sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 comprising the amino acid sequence of HGNFGNSYVSWFAY (SEQ ID NO: 18); a VL CDR1 comprising the amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 comprising the amino acid sequence of GTNCRAP (SEQ ID NO: 20); and a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 21).

[0507] 108. The multispecific polypeptide construct of embodiment 106 or embodiment 107, wherein the anti-CD3 dsFv comprises:

[0508] a VH having the amino acid sequence of any one of SEQ ID NOs: 14 and 32-62, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14 and 32-62; and

[0509] A VL having the amino acid sequence of any one of SEQ ID NOs: 15 and 63-81, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 14 and 32-62.

[0510] 109. The multispecific polypeptide construct of any one of embodiments 106 to 108, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15.

[0511] 110. The multispecific polypeptide construct of any one of embodiments 102 to 104, wherein the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44 and the amino acid sequence of SEQ ID NO: 72.

[0512] 111. The multispecific polypeptide construct of any one of embodiments 1 to 109, wherein the multispecific polypeptide construct is coupled to an agent.

[0513] 112. The multispecific polypeptide construct of embodiment 111, wherein the agent is a therapeutic agent, an anti-tumor agent, a toxin or a fragment thereof, a detectable portion or a diagnostic agent.

[0514] 113. The multispecific polypeptide construct of embodiment 112, wherein the agent is coupled to the multispecific polypeptide construct via a linker.

[0515] 114. A polynucleotide encoding the multispecific polypeptide construct of any one of embodiments 1 to 113.

[0516] 115. A polynucleotide encoding any polypeptide chain of the multispecific polypeptide construct of any one of embodiments 1 to 113.

[0517] 116. A polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of a multispecific construct of any one of embodiments 1 to 115 and a second nucleic acid sequence encoding a second polypeptide of the multispecific construct, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping.

[0518] 117. The polynucleotide of embodiment 116, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter.

[0519] 118. The polynucleotide of embodiment 116 or embodiment 117, wherein the multi-specific polypeptide construct comprises a third polypeptide chain, and the polynucleotide further comprises a third nucleic acid encoding the third polypeptide of the multi-specific construct.

[0520] 119. A polynucleotide as described in embodiment 118, wherein the third nucleic acid is separated from the first nucleic acid and / or the second nucleic acid by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jumping, and / or the third nucleic acid sequence is operably linked to the same promoter as the first nucleic acid sequence and / or the second nucleic acid sequence.

[0521] 120. The polynucleotide of any one of embodiments 116 to 119, wherein the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A or F2A.

[0522] 121. A vector comprising the polynucleotide of any one of embodiments 114 to 120.

[0523] 122. The vector of embodiment 121, which is an expression vector.

[0524] 123. The vector of embodiment 121 or 122, which is a viral vector or a eukaryotic vector, optionally wherein the eukaryotic vector is a mammalian vector.

[0525] 124. A cell comprising one or more polynucleotides of any one of embodiments 114 to 120 or one or more vectors of any one of embodiments 121 to 123.

[0526] 125. The cell of embodiment 124, wherein the cell is recombinant or isolated.

[0527] 126. The cell of embodiment 125, wherein the cell is a mammalian cell.

[0528] 127. The cell of embodiment 126, wherein the cell is a HEK293 or CHO cell.

[0529] 128. A method for producing a multi-specific polypeptide construct, the method comprising introducing into a cell one or more polynucleotides as described in any one of embodiments 114 to 120 or one or more vectors as described in any one of embodiments 121 ...

Claims

1. A multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3 binding region, wherein: The first component and the second component are coupled by a linker, wherein the Fc region is located at the N-terminus of the CD3 binding region; and One or both of the first component and the second component comprises an antigen binding domain that binds a tumor associated antigen (TAA).

2. The multispecific polypeptide construct of claim 1, wherein the CD3 binding region binds CD3 (CD3ε).

3. The multispecific construct of claim 1 or claim 2, wherein the antigen binding domain is located amino-terminally relative to the Fc region of the multispecific polypeptide construct and / or carboxy-terminally relative to the CD3 binding region.

4. The multispecific polypeptide construct of any one of claims 1 to 3, wherein the first component comprises a first antigen binding domain and the second component comprises a second antigen binding domain, wherein each of such antigen binding domains binds a tumor associated antigen (TAA).

5. The multispecific polypeptide construct of claim 4, wherein the first antigen binding domain is located amino-terminal to the Fc region of the multispecific construct, and the second antigen binding domain is located carboxyl-terminal to the CD3 binding region of the multispecific construct.

6. A multispecific polypeptide construct, wherein the multispecific construct comprises, from N-terminus to C-terminus: a first antigen binding domain that binds to a tumor-associated antigen (TAA); Immunoglobulin Fc region; connector; a CD3 binding region that binds CD3 (CD3ε); and The second antigen binding domain binds a tumor associated antigen (TAA). 7 . The multispecific polypeptide construct of claim 1 , wherein the Fc region is the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4, or an immunologically active fragment thereof.

8. The multispecific polypeptide construct of any one of claims 1 to 7, wherein the Fc region comprises: a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4; or A polypeptide comprising the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

5.

9. The multispecific polypeptide construct of any one of claims 1 to 8, wherein the Fc region is a heterodimeric Fc region.

10. The multispecific polypeptide construct of claim 9, wherein one or both Fc polypeptides of the heterodimeric Fc region comprise at least one modification to promote heterodimerization compared to a polypeptide of the homodimeric Fc region, optionally compared to the Fc polypeptide set forth in SEQ ID NO: 1 or an immunologically active fragment thereof.

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